A restriction endonuclease map of Streptomyces phage VWB

Summary A physical map of the actinophage VWB has been constructed using the restriction endonucleases BglII, ClaI, EcoRI, EcoRV, HindIII, KpnI and SphI. Phage VWB, genome size 47.3 kb, propagates on Streptomyces venezuelae, and it can also lysogenise this species. The three BglII-generated fragments of VWB DNA were cloned in pBR322, and subsequently mapped. In this manner the restriction map of the VWB phage genome was constructed.Abbreviations dam DNA adenine methylase activity - kb kilobase pairs - :: novel joint     

Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction

Summary We investigated whether the efficiency of transformation of plant cells by Agrobacterium tumefaciens during cocultivation is limited by the properties of the plant cells or by the infecting bacteria.Therefore, tobacco protoplasts were infected by cocultivation with two different agrobacteria strains carrying Ti plasmids with distinguishable T-DNAs. These T-DNAs cotransform plant cells at a frequency equal to the product of their independent transformation frequencies, which indicates that all plant cells are equally competent. On the other hand, when these T-DNAs are located on the same Ti plasmid vector within one bacterial strain, the cotransformation frequency is significantly higher than the product of the single transformation frequencies. We interpret these results to indicate that transformation is limited more by the establishment of effective bacteria/plant cell interaction than by (i) the process of DNA integration and (ii) by the number of plant cells capable of being transformed by Agrobacterium. We found that most plant cells are transformed by only one or a few agrobacteria. Analysis of the number of T-DNA copies in these clonally transformed lines indicates amplification of the original, infecting T-region copy.     

The use of graft copolymers to inhibit the adhesion of bacteria to solid surfaces

Twelve graft copolymers have been evaluated for their ability to prevent the adhesion of bacteria to substrata. The copolymers had polyethylene glycol (PEG) side-chains (‘teeth’) and a backbone that was either uncharged, acidic, basic or amphoteric. The copolymers were adsorbed onto glass, stainless steel and hydroxyapatite substrata, and 2-hpetri-dish adhesion experiments performed with bacteria isolated from marine (Pseudomonas sp. NCMB 2021), paper mill (S. marcescens NCIB 12211) and oral (S. mutans NCTC 10449) environments. The copolymers containing the most charged groups in the backbone had the most significant effect on bacterial adhesion levels, with anti-adhesive effects up to 99% achieved. An amphoteric copolymer (Compound 12) on glass, and acidic copolymer (Compound 11) on stainless steel and hydroxyapatite gave the most impressive anti-adhesive effects. These copolymers had non-specific bacterial anti-adhesive properties.It is proposed that the graft copolymers adsorbed onto hydrophilic surfaces via their charged backbone in such a way that the PEG side-chains were pointing out into the aqueous phase, and it was this orientation that was responsible for the observed anti-adhesive effect.     

Nocturnal parasitism of moth eggs by Trichogramma wasps

Parasitoid wasps of the genus Trichogramma are used worldwide as biological control agents against lepidopteran pests. Trichogramma wasps develop inside eggs of a wide range of host species, most of them moths. They are generally considered as diurnal insects. Here, we investigated whether Trichogramma wasps can also successfully parasitise host eggs at night under controlled laboratory conditions. Eggs of the moth Ephestia kuehniella were offered under dark conditions (scotophase) to females of Trichogramma brassicae and Trichogramma evanescens either from 9:00 PM to 9:00 AM or from 11:00 AM to 5:00 PM at four different temperatures (5°C, 10°C, 15°C and 20°C). Both species are known to parasitise E. kuehniella eggs in the photophase during daytime. The results show that T. brassicae did not parasitise eggs in the scotophase at night and only very few in the artificially induced scotophase during daytime from 10°C to 20°C. In contrast, T. evanescens parasitised more eggs in the dark both at night and artificially induced scotophase during daytime. Parasitism in the scotophase already started at 5°C, with more eggs being parasitised and more offspring being produced at higher temperatures. T. evanescens displayed higher parasitism activity in the induced scotophase during daytime than in the scotophase at night. The present study suggests that Trichogramma are capable of successfully parasitising host eggs at night, even at low temperatures, but that nocturnal activity with respect to parasitism varies between wasp species.     

A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting ß-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from

ABSTRACT: BACKGROUND: D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a beta-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. RESULTS: In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52[DEGREE SIGN]C; however, it exhibited over 60% of maximum activity at 30[DEGREE SIGN]C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50[DEGREE SIGN]C. In this study, a recombinant Pichia pastoris yeast strain secreting beta-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting beta-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization of D-glucose and a 30% conversion of D-galactose to D-tagatose. CONCLUSIONS: The method developed for the simultaneous hydrolysis of lactose, utilization of D-glucose and isomerization of D-galactose using a P. pastoris strain secreting beta-D-galactosidase and recombinant L-arabinose isomerase seems to offer an interesting alternative for the production of D-tagatose from lactose-containing feedstock.