Transformation of Bacillus subtilis by electroporation

Summary Optimal conditions for the transformation of Bacillus subtilis by electroporation were achieved. Frequencies of greater than 10⁵ transformants/μg of plasmid DNA were obtained for a number of strains and plasmids. Increased transformation efficiency of mini-prep DNA from B. subtilis and Escherichia coli was obtained after microdialysis.     

A highly efficient electroporation system for transformation of Bacillus licheniformis

Summary Transformation of intact cells of Bacillus licheniformis 5A24 with plasmids pLM6 (2.8 kb), pC194 (2.9 kb) and pCP49 (7.1 kb) by electroporation resulted in 1.5 × 10⁶, 1.2 × 10⁶ , and 5.2 × 10⁴ transformants per μg DNA, respectively. Transformation was possible with plasmid DNA, which was religated after restriction endonuclease digestions. In addition, evidence is presented that indicates that B. licheniformis possesses DNA restriction and modification systems.     

Silver toxicity to ferrous iron and pyrite oxidation and its alleviation by yeast extract in cultures ofThiobacillus ferrooxidans

Summary Ferrous-ion oxidation byThiobacillus ferrooxidans was inhibited by 10^–6 M Ag⁺ while a slight inhibition of growth was apparent with 10^–7 M Ag⁺. The threshold toxic concentration was the seme for four different test strains. While prolonged lag phases resulted from culture exposure to Ag⁺, Fe²⁺ oxidation rates after the onset of growth showed little variation under these conditions. Yeast extract (0.02%) partially alleviated the toxicity of silver-ion by reducing the lag periods. Pyrite oxidation byT. ferrooxidans and mixed cultures of acidophiles was tested at 8.3×10^–7 to 8.3×10^–5 M Ag⁺. Strong inhibition was apparent at 8.3×10^–5 M Ag⁺ and little to no inhibition was observed at 8.3×10^–7 M Ag⁺.     

Growth and survival of Bacillus cereus in mageu,a sour maize beverage

The growth and survival of Bacillus cereus, a known pathogen commonly found in cereals, during lactic acid fermentation of mageu, a sour maize beverage, was studied. In the mageu base inoculated with both the starter culture and B. cereus, the acidity developed to pH 4.00 and 0.10% titratable acidity after 24 h; the growth of B. cereus was reduced from 10⁶ c.f.u./ml to 10² c.f.u./ml within 24 h; after the first 6 h of fermentation, the rate of inhibition of B. cereus was correlated to the rate of decrease in pH (r = 0.85, p < 0.05); the redox potential (E_h) decreased from 463 to 149 mV within the first 12 h. The control mageu base to which neither starter nor lactic acid was added, had a pH of 6.50, titratable acidity of 0.015% and lowest E_h of 244 mV. In the mageu base to which lactic acid and B. cereus were added, the pathogen was inhibited to < 10¹ c.f.u./ml. The B. cereus in the mageu base to which no starter culture nor lactic acid was added, grew to over 10⁷ c.f.u./ml after 12 h. The decrease in E_h seemed to have no inhibitory effect on the growth and survival of B. cereus. No strains of lactic acid bacteria were found to produce bacteriocins antagonistic to B. cereus. Low pH and acidity were found to be the major factors inhibiting growth of B. cereus in mageu.     

Optimization of a medium for a high yield production of spore-crystal preparation ofBacillus thuringiensis effective against the Egyptian cotton leaf wormSpodoptera littoralis Boisd.

Summary The continuous culture (chemostat) technique was used for the optimization of a medium which supported the production of a high yield spore-crystal preparation ofBacillus thuringiensis (isolate Bt 24) (a maximum of 4 × 10⁹spores/ml) on a pilot-scale. The preparation demonstrated a high insecticidal activity against second-instar larvae ofSpodoptera littoralis Boisd.     

Evidence for a magnesium pump in Bacillus cereus T

Unlike Escherichia coli, Bacillus cereus T appears to accumulate Mg(2+) in its cell sap against a concentration gradient. Over a range of Mg(2+) in the growth medium from 5 x 10(-5) to 1.35 x 10(-2)m, the concentration of Mg(2+) in the cell sap of B. cereus T was maintained at about 6 x 10(-3)m, and ribosome-bound Mg(2+) and spermidine, as well as the spermidine concentration in the cell sap, appear to be unaffected by the concentration of Mg(2+) in the growth medium. Inhibition of growth of E. coli by streptomycin is progressively reversed by increasing the concentration of Mg(2+) in the growth medium above 5 mm. The finding that similar increases of Mg(2+) in the growth medium did not reverse the inhibition of B. cereus T is also consistent with the conclusion that B. cereus T, unlike E. coli, accumulates Mg(2+) to a constant concentration in its cell sap.     

Optimum tissue culture conditions for selection of resistance to Phytophtora cinnamomi in pine callus tissue

The present experimentation compared the best nutrient medium, temperature, and growth hormones for callus induction and growth of various pine species from different seed sources with their effect on growth of Phytophthora cinnamomi. Callus tissues maintained on a modified Murashige and Skoog medium with 10^–5M 2,4-D at 26°C in the dark optimized the expression of differential resistance when inoculated with hyphae of P. cinnamomi. High concentration of 2,4-D (5×10^–5M) inhibited growth of P. cinnamomi.Abbreviations AL loblolly pine-Alabama - PL South Carolina - AS shortleaf pine-Alabama - CS Georgia - AV Virginia pine-Alabama     

Fusion of Agrobacterium and E. coli spheroplasts with Nicotiana tabacum protoplasts — Direct gene transfer from microorganism to higher plant

Spheroplasts of Agrobacterium tumefaciens strains and E. coli were fused with protoplasts of Nicotiana tabacum. Fusion products were cultured in the presence of antibiotics to eliminate remaining bacterial spheroplasts. On hormone free medium, tobacco protoplasts treated with wild type Agrobacterium-strains formed colonies with an average frequency of 10^–4. Opine synthesis was detected in the tissues. Some calli derived from protoplasts treated with A. tumefaciens C58C1pRi15834 formed typical hairy roots. Kanamycin resistant calli were obtained after fusion with A. tumefaciens containing pLGVTi23 neo (frequency=10^–3). Fusion of E. coli spheroplasts containing a virulent pTiB6S3::RP4 co-integrate with tobacco protoplasts yielded two hormone independent growing calli producing octopine out of 10⁵ microcalli.Abbreviations PEG Polyethylene glycol - PVA Polyvinyl alcohol     

Bacteria of the genus Bacillus have a hydrolase stereospecific to the D isomer of benzoyl-arginine-p-nitroanilide.

A stereospecific enzyme activity capable of cleaving the amide bond of the synthetic substrate N-benzoyl-D-arginine-p-nitroanilide (D-BAPA) has been found in all aerobic and anaerobic members of the family Bacillaceae tested by us. Cells of nonsporeforming gram-positive or gram-negative bacteria contain a hydrolase activity stereospecific to N-benzoyl-L-arginine-p-nitroanilide. The D-BAPA-hydrolyzing enzymes (D-BAPAases) of mid-logarithmic-phase cells of Bacillus subtilis 168 and B. cereus T were compared. These enzymes had the same molecular weight of approximately 66,000 in gel filtration and the same electrophoretic mobility after electrophoresis on polyacrylamide gels. The D-BAPAases of B. subtilis 168 and B. cereus T differed in the effect of inhibitors on enzymatic activity. While both hydrolases were inhibited by tosyl-L-lysine chloromethyl ketone and tosyl-L-arginine-methyl ester as well as leupeptin, only the D-BAPAase of B. cereus T was inhibited by p-chloromercuribenzene sulfonic acid. The D-BAPAases of B. subtilis and B. cereus T had a Michaelis constant for D-BAPA of 2.9 x 10(-5) M and 1.4 x 10(-4) M, respectively. D-BAPAase is an intracellular enzyme localized in the protoplast (80 to 90% in soluble form in the cytoplasm). The ability to cleave D-BAPA is suggested as an additional chemotaxonomic characteristic of sporeforming bacteria of the genera Bacillus and Clostridium.     

Transformation of Bacillus megaterium by electroporation

Summary Plasmidic DNA was introduced into intact cells of Bacillus megaterium by electroporation. The procedure showed an efficiency of 10³ transformants g^–1 DNA.     

L-Lysine production by mutants of Bacillus licheniformis

Summary A bacterium able to grow at 46°C was isolated from soil and identified asBacillus licheniformis. L-Lysine producing mutants were derived from the bacterium by the introduction of resistance to S-(2-amino)-ethylcysteine (thialysine) and auxotrophy.One of the mutants, HBR-2 (Thialysine^r, Leu^–, Homoserine^–), produced L-lysine at a concentration of 30 mg/ml in a molasses medium containing 10% reducing sugar.     

Metabolism of progesterone and testosterone by a Bacillus sp.

Microbial transformations by a Bacillus sp. were employed as a means of preparing potentially important derivatives of progesterone and testosterone. Each microbial metabolite was subjected to structure elucidation employing ¹H and ¹³C nmr, mass spectral and cd analysis. Hplc was used for the determination of the percentages of the metabolites formed. The progesterone metabolites were characterised as 14-hydroxy-4-pregnene-3,20-dione (II), 14-hydroxy-5 α -pregnane-3,6,20-trione (III)., 11 α — hydroxy-5 α — pregnane-3, 6,20-trione (IV) and 11 α, 14-dihydroxy-4-pregnene-3,20-dione (V). The testosterone analogs were identified as 4-androstene-3,17-dione (VII), 17 β-hydroxy-5 α -androstene-3,6-dione (VIII), 14-hydroxy-4-androstene-3,17-dione (IX) and 14, 17 β-dihydroxy-4-androsten -3-one (X)₁. The availability of the metabolites enabled complete elucidation of their ¹³C nmr spectra.     

Somatic embryogenesis and organogenesis in callus cultures of a tree legume — Albizia richardiana King

Hypocotyl explants of 1 and 10 mm lengths were excised from 12-day-old in vitro-grown seedlings of Albizia richardiana. The larger pieces, after 40 days of culture, developed shoots along with green calli on B₅ + BAP (10^–7–10^–5M), while the smaller segments produced only green calli on B₅+BAP (10^–7–10^–4M) medium. Some of the green calli turned morphogenic and started producing somatic embryos with the 2nd sub-culture and shoots from 7th sub-culture onwards. Calli retained the morphogenic potential even after repeated sub-culturing for over two years. The number of embryos in an embryogenic culture varied from 2 to 20 per callus mass of 5–6.5 cm³. Sucrose at the 2% level in MS medium was optimal for embryogenesis while 4% was optimal for shoot bud differentiation. Higher levels of sucrose (6–10%) caused browning of green calli and also inhibited differentiation into embryos and shoot buds. By selective sub-culturing of 0.1 cm³ pieces of embryogenic calli on MS+10^–5M BAP, 46% of the cultures produced somatic embryos. The latter germinated into plantlets on Knop's medium.Abbreviations BAP 6-benzylaminopurine - B₅ Gamborg et al., 1968 medium - IAA Indole-3-acetic acid - MS Murashige and Skoog's (1962) medium     

Occurrence of cyclosporins and cyclosporin-like peptolides in fungi

Apart from 17 previously listed fungal taxa producing cyclosporin A and its natural congeners B to Z, several additional and taxonomically diverse strains producing the single novel component [Thr², Leu⁵, Leu¹⁰]cyclosporin or cyclosporin-like peptolides (eg SDZ 214-103=[Thr², Leu⁵, D-Hiv⁸, Leu¹⁰]cyclosporin) have recently been described. We report here the isolation of two further and novel cyclosporins, [Thr², Leu⁵, Ala¹⁰]cyclosporin (2) and [Thr², Ile⁵] cyclosporin (3), from strains classified asAcremonium luzulae (Fuckel) W Gams andLeptostroma anamorph ofHypoderma eucalyptii Cooke & Harkn, respectively. In both new strains the usual pattern of cyclosporins A to Z is not found. The structure elucidations of2 and3 are based on NMR spectroscopy, and biological data (immunosuppressive activity, cyclophilin-binding affinity and antifungal effects) are presented.     

Biomass production from the green algae Chlorella vulgaris and Ankistrodesmus braunii cultured heterotrophically

Summary Ankistrodesmus braunii and Chlorella vulgaris were cultured heterotrophically under various operating conditions. The maximum rate of biomass production was 900 and 900 mg L^-1 d^-1 by C. vulgaris and 1000 and 700 mg L^-1 d^-1 by A. braunii in the light and dark, respectively. This indicates that these algae could produce in excess of 1530 dry weight tonnes ha^-1 y^-1 which is 10–20 times higher than the maximum production levels in the literature.     

Spores of microorganisms

1. (1)
   Аланин-2-¹⁴C и³⁵S-метионин требования клеток Bacillus cereus и Bacillus мегатерий не увеличивают в течение sporulation. Существу ет, однако, Увеличение включени я³⁵S-цистеина.     
   
   

Production of virus resistant and insect tolerant transgenic tobacco plants

The cucumber mosaic virus coat protein (CMV-CP) gene and a modified Bacillus thuringiensis -endotoxin (Bt toxin) gene were cloned into plant expression vector pE3. Tobacco (Nicotiana tabacum cv. G28) leaf discs were transformed with Agrobacterium tumefaciens A12 carrying recombinant pE14. Transgenic r₀ and R₁ tobacco plants expressing CMV-CP and Bt toxin genes were protected from CMV infection as well as feeding damage of Manduca Sexta (tobacco hornworm) larvae. These results demonstrate that it is feasible to breed new cultivars with multiple resistances via genetic engineering.Abbreviations CMV cucumber mosaic virus - Bt toxin Bacillus thuringiensis -endotoxin - CaMV cauliflower mosaic virus - NOS nopaline synthase - Kan Kanamycin - Spe spectinomycin - Carb Carbenicillin     

Effect of cinnamoyl putrescines on in vitro cell multiplication and differentiation of tobacco explants

Hydroxycinnamoyl putrescines promote the cell multiplication of leaf discs of a tobacco mutant, RMB₇, cultivatedin vitro on the Murashige and Skoog medium. This mutant never accumulates these molecules during its development and does not enter in floweirng. Maximal effect is obtained at 2.5·10^–4M. The same molecules inhibit bud formation ofNicotiana tabacum var. Xanthi nc, at 5·10^–5 M but promote callus formation. From 10^–4 M to 5·10^–3 M they strongly inhibit cell multiplication and bud formation without toxic effect. Their possible role in plant metabolism is discussed.Abbreviations IAA indole 3-acetic-acid - BA benzyladenine     

Nucleosides,I Ribosylation of 8-Substituted Theophylline Derivatives

Abstract

The attempted ribosylation reaction of 8-nitro-theophylline (2) with 1-o-acetyl-2, 3, 5-tri-o-benzoyl-D-ribo-furanose (5) failed to give any nucleoside product, whereas the reaction of 8-chlorotheophylline (3) with 5 afforded the 8-chloro-7-(2,3,5-tri-o-benzoyl) β-D-ribofuranosyltheophylline (6) in good yield. The product 6 reacted with benzylamine producing the 8-benzylamino-7-(2, 3, 5-tri-O-benzoyl) β-D-ribo-furanosyltheophylline (10), which could also be synthesised by ribosylation of 8-benzylaminotheophylline (8) with 5. Debenzoylation of 6 and 10 gave the corresponding 7-β-D-ribofuranosyltheophylline nucleosides (7) and (11), respectively. Compound 7 could be converted into 11 by reaction with benzylamine. The newly synthesised compounds have been characterised by elemental analysis, ¹H-NMR and UV spectra.     

Stimulatory effects of prostaglandin E1 on rat anterior pituitary cyclic amp and luteinizing hormone release

The effect of prostaglandin E₁ (PGE₁) on rat anterior pituitary cyclic AMP accumulation and luteinizing hormone (LH) release was studied both in vivo and in vitro. Addition of PGE₁ to incubation medium over a concentration range of 10^-6 to 10^-4 M produced a graded increase in pituitary cyclic AMP. At the lowest concentration (10^-6 M) there was no significant increase in LH release, but proportional increments in LH release were seen with increasing concentrations of PGE₁.Ten minutes after intravenous administration of 5 μg of PGE₁ to adult male rats, pituitary cyclic AMP was substantially increased while serum LH levels were not changed. Administration of a higher dose of PGE₁ (20 μg) produced a greater increase in pituitary cyclic AMP; and, at this dose serum LH was significantly increased. These results suggest that the PGE₁ effect on LH release is mediated by the adenyl cyclase — cyclic AMP system.