Conjugal transfer of Streptococcal antibiotic resistance plasmids intoClostridium acetobutylicum

Summary Streptococcal plasmids pAM1, pVA797, pVA797Tn917 and pAM610 were transferred or mobilized toClostridium acetobútylicum fromStreptococcus sanguis,S. faecalis andS. lactis donors.Clostridum transconjugants were able to retransfer pAM1 and pVA797 to a plasmid-freeS. lactis recipient.     

Characterization of two β-glucosidase genes fromClostridium thermocellum

Summary Two -glucosidase genes, designatedbglA andbglB, were isolated from a gene bank ofClostridium thermocellum DSM 1237. The coding sequences forbglA andbglB were located on non-homologous DNA fragments of 3.2– and 3.4-kb, respectively. Both genes direct inEscherichia coli the synthesis of cytoplasmic -glucosidases, which differ with respect to substrate specificity and temperature profile. The properties of thebglA-encoded -glucosidase A closely resemble that of a -glucosidase previously isolated fromC. thermocellum cultures.     

Fermentation of starch to ethanol by a complementary mixture of an amylolytic yeast andSaccharomyces cerevisiae

Summary Synergistic coculture of an amylolytic yeast (Saccharomycopsis fibuligera) andS. cerevisiae, a non-amylolytic yeast, fermented unhydrolyzed starch to ethanol with conversion efficiencies over 90% of the theoretical maximum. Fermentation was optimal between pH 5.0 to 6.0. Using a starch concentration of 10% (w/v) and a 5% (v/v) inoculum ofS. fibuligera, increasingS. cerevisiae inoculum from 4% to 12% (w/v) resulted in 35–40% (w/v) increase in ethanol yields. Anaerobic or limited aerobic incubation almost doubled ethanol yields.     

Resistance of bacterial film to contamination during use as inoculum in repeated batch fermentation

Summary AnEscherichia coli strain constitutive for -galactosidase was immobilized onto cotton cloth. The resultingE.coli film was used as a resident inoculum in repeated batch fermentations for 30 days in the presence ofBrevibacterium ammoniagenes added as a contaminant. Analysis of -galactosidase production shows that contamination did not decrease the capacity of the film to generateE.coli cells, or decrease theE.coli population on the film.     

Production and conversion of chitosan with cultures of Mucor rouxii or Phycomyces blakesleeanus

Summary Highly deacetylated chitosan was accumulated in the mycelia ofMucor rouxii orPhycomyces blakesleeanus. These cultures also effected the deacetylation of the chitin ofAspergillus niger mycelium into chitosan. After 96 hours of incubation with these cultures the degree of acetylation of commercial crab shell chitosan was reduced from 25.0% to values between 4.3 and 8.6%. The potential exists for the production of chitosans with tailored physico-chemical properties from waste chitin.     

Growth of selected fungi on leaf-protein supernatant

Summary Some 19 strains ofAspergillus niger,A. oryzae, andPaccilomyces spp. are tested for their ability to grow on the supernatant remaining after the expressed juice from sugar beet tops and meadow grass has been heat-treated to precipitate crude leaf protein, and supplemented as required by glucose or ammonium sulphate. With effective strains ofA. niger,A. oryzae,P. elegans orP. variotii and an optimized carbohydrate/nitrogen ratio, over 70% of the organic content of the supernatant is rapidly converted into mycelial biomass of high protein content.     

Regulation of mycotoxin production by Fusarium graminearum: Complementation effects between two mutant types

Summary Starting from anileu auxotroph ofFusarium graminearum producing high levels of the mycotoxin zearalenone, selection after UV irradiation gave low-producing mutants of essentially normal morphology,zea,ileu. Heterokaryons betweenzea,ileu strains and an auxotrophic strainlz,inos derived from the lazy morphological mutant ofFusarium graminearum, which has abnormal morphology and also produces little or n zearalenone, produced significant levels (over 50% of the wild-type level) of mycotoxin. The observation confirms views as to the regulatory nature of thelazy mutation.     

High level extracellular secretion of thermostable α-amylase ofBacillus stearothermophilus inEscherichia coli

Summary Bacillus stearothermophilus BR135 (ATCC 29609)amy gene was cloned in pBR322 from its plasmid DNA and was subcloned in a vector useful both forB. subtilis andE. coli.E.coli HB101 harboring the plasmid pSS099 when grown in L medium in presence of 5. g/ml chloramphenicol produces 70 units/ml of extracellular -amylase. This is nearly twice that ofE.coli cells harboring pSSO76, a plasmid havingamy ofB.stearothermophilus BR135 atHindIII site of pBR322. Characteristically the protein was a 58 kd protein and cross reacted with antiserum developed against purified -amylase of BR135.     

Towards a classification ofE. coli ribosomal proteins: A hypothetical ‘small ribosome’ as a primitive protein-synthesizing apparatus

Homologies were searched among the published primary sequences of 51E. coli ribosomal proteins, partly by eye and partly by computer-assisted methods. By employing Moore and Goodman's alignment statistics for evaluating homology levels, 33 out of these 51 ribosomal proteins has been classified into 9 homology groups, some of which being yet tentative and remaining to be further analyzed. Taking it into consideration that most ribosomal protein genes are clustered atstr-stc region,rif region and several other regions, these results strongly suggest that most or all of the contemporary ribosomal proteins must have evolved by repeated gene duplications ofvery few (oronly one) primitive ancestral ribosomal protein gene(s). Thus it is most reasonable to propose that a small ribosome consisting of very few (or only one) ribosomal protein(s) must have existed as a primitive protein-synthesizing apparatus.     

Studies of dotted,a regulatory element in maize

Summary Dotted (Dt) is the regulatory element of a two-unit controlling system in maize. Dt causes the inherited change from the recessive a (colorless) to its dominant allele, A (anthocyanin production), during the development of the stalk, leaves, and endosperm. The mutation events are observed as sectors of color in an anthocyaninless background.Since its discovery over 40 years ago, Dt has always been found in the terminal knob of the short arm of chromosome 9. This is puzzling because controlling and regulatory elements in general are not located permanently, but change positions (transpose) within the chromosomal complement. To resolve this seeming discrepancy, transpositions were looked for in a homozygous a Dt stock. Because the frequency of aleurone mutations is exponentially related to Dt dosage, a Dt transposition would result in a greatly increased number of dots if the egg or sperm nucleus contained both the transposed Dt and the Dt remaining on chromosome 9. A total of 6 transposed Dt's (Dt-T) were recovered in this manner. Dt-TA was found linked to the gene Y (yellow endosperm) of chromosome 6. Dt-TB no longer showed linkage with yg2 of chromosome 9, but remains unlocated (the original Dt in this stock is separated from yg2 by 6 or 7 cross-over units.). The remaining transpositions (C-F) assorted independently of Dt on chromosome 9.The transposed Dt's had the same effect as Dt on the frequency and timing of aleurone mutations. An increase in transposition frequency and losses of Dt-T's was characteristic of several of the transposed Dt's. Dt-T's B-F transposed so frequently that testcross ratios of 71 (three Dt' s) and 15 1 (four Dt' s) were observed. No secondary transpositions or losses of Dt-TA were detected. Thus, Dt-TA resembles the original Dt with regard to its transposition frequency and stability.Journal Paper No. J-8333 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 1880.     

Synthesis of novel sugars,oligoglucosyl-inositols,and their growth stimulating effect forBifidobacterium

Summary Novel sugars, oligoglucosyl-inositols, which were synthesized using CGTase fromBacillus ohbensis, stimulated the growth ofBifidobacterium. The enzyme catalyzed transglucosylation from -1,4-maltodextrin (donor) tomyo-inositol (acceptor). Of donors examined, -cyclodextrin gave superior oligoglucosyl-inositol yield of 56.6% (w/w) based on the conversion ratio of incubated inositol. Maltosyl-inositol stimulated growth ofB. adolescentis by 194% when compared with glucose.     

A negative selection scheme for tobacco protoplast-derived cells expressing the T-DNA gene 2

The amido hydrolase encoded by the T-DNA gene 2 catalyzes the conversion of indole-acetamide, -naphthalene acetamide, and other substrate analogues into the corresponding auxins. As a result, only gene 2-expressing protoplast-derived tobacco cells can grow in medium containing low concentrations (0.2–1 M) of -naphthalene acetamide as auxin precursor. However, in a mixture of SR1 and SR1, gene 2 ⁺ protoplast-derived cells, cross-feeding occurs and consequently no positive selection for gene 2 is obtained. A 100-times higher concentration of -naphthalene acetamide (between 30 and 300 M) provides a negative selection scheme. Only the tobacco cells expressing gene 2 are sensitive to the high naphthalene acetamide concentration and cannot grow to colonies, while cells lacking the gene 2 product regenerate calli even in mixed gene 2 ⁺ and gene 2 ^– cell populations. Thus, gene 2 might provide a unique biochemically defined marker to investigate mutations and gene inactivation.     

Polymerization of amino acids containing nucleotide bases

Summary We have prepared the nucleoamino acids 1-(3-amino, 3-carboxypropyl)uracil (3) and 9-(3-amino, 3-carboxypropyl)adenine (4) as (l)-enantiomers and as racemic mixtures. When3 or4 is suspended in water and treated with N,N-carbonyldiimidazole, peptides are formed in good yield. The products formed from the (l)-enantiomers are hydrolyzed to the monomeric amino acids by pronase.Attempts to improve the efficiency of these oligomerizations by including a polyuridylate template in the reaction mixture were not successful. Similarly, oligomers derived from the (l)-enantiomer of3 did not act as templates to facilitate the oligomerization of4.     

Secondary product formation by cultures of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes

Hairy root cultures of Beta vulgaris and Nicotiana rustica were established after roots were induced on plants following infection with Agrobacterium rhizogenes. The transformed cultures of B. vulgaris and N. rustica synthesised their characteristic secondary products, the betalain pigments and nicotine alkaloids respectively, at levels comparable with those of in vivo roots from the same variety. Betalains were entirely retained inside the root tissue. In contrast, a proportion of the nicotine alkaloids was secreted into the medium. The potential of this type of in vitro plant tissue culture for the production of valuable plant secondary products is identified and confirmed.     

Acetone-butanol-isopropanol production byClostridium beijerinckii (synonym,Clostridium butylicum)

Isopropanol is a product of the organism formerly known as Clostridium butylicum, which is now included in the speciesClostridium beijerinckii. We tested 52 strains ofC.beijerinckii for their ability to produce acetone,n-butanol, and isopropanol. The 32 butanol-producing strains may be divided into two groups based on whether isopropanol was produced. Isopropanol-producing cultures accumulated acetone only transiently.     

Fates and expression ofBacillus subtilis endo-β-1, 4-glucanase gene andAlcaligenes faecalis β-glucosidase gene introduced inEscherichia coli andBacillus cells

Summary Two kinds of cellulase genes coding for endo--1, 4-glucanase and -glucosidase, isolated fromBacillus subtilis andAlcaligenes faecalis respectively, were separately or combinedly put on a newly constructedEscherichia coli-Bacillus shuttle vector plasmid. When the recombinant plasmids having cellulase gene(s) were introduced intoE. coli orBacillus cells, drastic differences in fates and expression of the two genes were observed.     

In vitro plant regeneration of leguminous trees (Albizia spp)

Hypocotyl explants of three leguminous forest tree species, Albizia amara, A. lucida and A. richardiana, have differentiated shoot buds on B₅ basal medium. Maximum number of shoots per explant developed on basal medium augmented with 2,4-D (0.1 M) in A. amara (2) and BA (10 M) for both A. lucida (2) and A. richardiana (1.6). Higher concentrations of auxins in the medium, in general, enhanced rooting and callusing but cytokinins promoted the growth of green calli. BA enchanced the differentiation of shoots in the three species. The in vitro grown shoots of A. amara and A. richardiana, after subculturing on B₅+1 M IAA developed roots (up to 30–40%). These plants have been successfully transferred to the field.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - BM Gamborg's B₅ medium with 0.9% agar+3% sucrose - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kn Kinetin - NAA -naphthaleneacetic acid     

Plant regeneration from callus cultures of Durum and emmer wheat

Callus cultures were initiated from isolated mature embryos of Triticum turgidum L. Thell ssps durum and dicoccum on a basal medium supplemented with 2,4-D, 2,4,5-Cl₃POP or 2,4-D+CM. Shoot bud regeneration was observed on 2,4,5-Cl₃POP medium. In both the cultivars of durum, further development of shoot buds occurred on transfer of tissues to basal medium whereas in dicoccum basal medium supplemented with coconut milk or coconut milk with NAA (0.2 mg/l) was necessary. The regenerated shoot buds were induced to root on basal medium supplemented with NAA. The in vitro obtained plants were transferred to soil and successfully grown to maturity. Chlorophyll variants were observed among the regenerated plants of dicoccum.Abbreviations BA benzyladenine - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,iP 6---dimethylallylamine purine - IAA indoleacetic acid - NAA -naphthalene acetic acid - Kn kinetin - 2,4,5-Cl₃POP 2,4,5-trichlorophenoxypropionic acid - MS modified Murashige and Skoog's medium - RH relative humidity - Z zeatin     

Microbial asymmetric oxygenation of an organometallic sulfide

Summary Penicillium frequentans IFO 5692 oxidized enantioselectively (methylthiomethyl)ferrocene (1) to (R)-(–)-(methylsulfinylmethyl)ferrocene (2) (98 %e.e.) andCorynebacterium equi IFO 3730 to (S)-(+)-2 (69 %e.e.).     

Somatic hybrids between Solanum brevidens and Solanum tuberosum: Expression of a late blight resistance gene and potato leaf roll resistance

Hexaploid somatic hybrids resulting from mesophyll protoplast fusions between Solanum brevidens Phil., PI 218228, and Solanum tuberosum L., PI 203900 were tested for late blight resistance using two races of Phytophthora infestans Monte., de Bary. The S. tuberosum parent was a late blight differential possessing the R4 gene which confers resistance to race 0. The S. brevidens parent is resistant to potato leaf roll virus. Inoculations with both compatible (race 1.3.4.5) and incompatible (race 0) races of P. infestans clearly demonstrated the expression of the late blight resistance gene in all of the hybrid progeny tested. Most of the hybrids tested were also resistant to potato leaf roll virus (PLRV), indicating that the S. brevidens genes for PLRV resistance were present and expressed.