Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1

The Pseudomonas aeruginosa protein PtxS negatively regulates its own synthesis by binding to the upstream region of its gene. We have recently identified a 14 bp palindromic sequence within the ptxS upstream region as the PtxS operator site (OP1). In this study, we searched the P. aeruginosa genomic sequence to determine whether this 14 bp sequence exists in other regions of the P. aeruginosa chromosome. Another PtxS operator site (OP2) was located 47 bp downstream of ptxS. DNA gel shift experiments confirmed that PtxS specifically binds to a 520 bp fragment that carries OP2. The DNA segment 3' of OP2 contains four open reading frames (ORF1-ORF4), which code for 29, 32, 48 and 35 kDa proteins respectively. The molecular weight of the products of ORFs 2 and 3 were confirmed by T7 expression experiments. Computer analyses suggest that ORF2 encodes an ATP-dependent kinase; ORF3, a transporter; and ORF4, a dehydrogenase. The predicted product of ORF1 showed no homology to previously identified proteins and contains all the conserved amino acids within the aldose 1-epimerase protein motif. Examination of the ptxs-ORF1 intergenic region (using promoter fusion experiments) showed that no potential promoter exists. An isogenic mutant defective in ORF1 was constructed in the P. aeruginosa strain PAO1. In contrast to its parent strain, the mutant failed to grow on a minimal medium in which 2-ketogluconate was the sole carbon source. Similarly, a previously constructed ptxS isogenic mutant of PAO1 did not grow in a minimal medium containing 2-ketogluconate as the sole carbon source. Furthermore, a plasmid carrying a fragment that contains ptxS and ORFs 1-4 complemented the defect of the previously described P. aeruginosa 2-ketogluconate-negative mutant. In the presence of 10 mM 2-ketogluconate, the in vitro binding of PtxS to a DNA fragment that carries either OP1 or OP2 was inhibited. These results suggest that: (i) ptxS together with the other four ORFs constitute the 2-ketogluconate utilization operon (kgu) in P. aeruginosa. Therefore, ORFs 1-4 were designated kguE, kguK, kguT and kguD respectively. (ii) PtxS regulates the expression of the kgu operon by binding to two operators (OP1 and OP2) within the operon; and (iii) 2-ketogluconate is the molecular inducer of the kgu operon or the molecular effector of PtxS.     

An improved method of photometric determination of cyclodextrin glucanotransferase activity

The method of spectrophotometric determination of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) activity with the use of phenolphthalein as a colored reagent has been improved. This technique includes an enzymatic reaction at 40°C for 60 min in 2% starch, with subsequent supplementation of the reaction mixture (0.5 ml) with the phenolphthalein reagent (3.0 ml) prepared in 0.1 M potassium carbonate buffer (pH 11.0) according to a special procedure, and measurement of the optical density of the obtained mixture at 553 nm. The activity was calculated using the exponential growth equation that connects a drop in the optical density and the degree of dilution of the enzyme. The described technique is suitable for working in a sufficiently broad range of specific activity of β-CGTase and does not require precise adjustment of the degree of dilution of solutions analyzed.     

The ultrastructure of somatic embryo development in pearl millet (Pennisetum glaucum; Poaceae)

The ultrastructure, morphology, and histology of somatic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Somatic embryogenesis was initiated from zygotic embryo explants cultured 8 d after pollination. Formation of a ridge of tissue began 3–4 d after culture (DAC) by divisions in the epidermal and subepidermal cells of the scutellum. Ridge formation was accompanied by a decrease in vacuoles, lipid bodies, and cell size, and an increase in endoplasmic reticulum (ER). Proembryonic cell masses (proembryoids) formed from the scutellar ridge by 10 DAC. Proembryoid cells had abundant Golgi bodies and ER while the amounts of lipids and starch varied. Somatic embryos developed from the proembryonic masses 13 DAC and by 21 DAC had all the parts of mature zygotic embryos. Although shoot and root primordia of somatic embryos were always less differentiated than those of zygotic embryos, scutellar cells of somatic and zygotic embryos had similar amounts of lipids, vacuoles, and starch. Somatic scutellar epidermal cells were more vacuolated than their zygotic counterparts. In contrast, somatic scutellar nodal cells were smaller and not as vacuolated as in zygotic embryos. Somatic embryogenesis was characterized by three phases of cell development: first, scutellar cell dedifferentiation with a reduction in lipids and cell and vacuole size; second, proembryoid formation with high levels of ER; and third, the development of somatic embryos that were functionally and morphologically similar to zygotic embryos.     

Phytochemical and flow cytometric analyses of Devil’s claw cell cultures

A cell suspension culture of Devil’s claw (Harpagophytum procumbens), a South African plant with high medicinal value, cultivated under submerged conditions showed stable growth and accumulated high amounts of biomass (18.2 g l⁻¹). Flow cytometry analyses of the suspension’s cell cycle kinetics showed that proportions of cells in G₀/G₁ and S phases varied insignificantly (between 69–76% and 9–13%, respectively) during the cultivation, while the proportion of G₂/M-phase cells increased until day 8 of cultivation, when the exponential phase of cell growth ended. Metabolite production in the culture was studied through simultaneous determination of three bioactive phenylethanoid glycosides (verbascoside, β-OH-verbascoside and leucosceptoside A) by high performance liquid chromatography. It was found that suspended Devil’s claw cells accumulated mainly verbascoside (517.3 mg l⁻¹), followed by leucosceptoside A (107.1 mg l⁻¹) and β-OH-verbascoside (80.3 mg l⁻¹). In addition, several fatty acids and β-sitosterol were identified in the cell suspension by gas chromatographic-mass spectrometry analysis. Comparison of the results with previously acquired data for Harpagophytum procumbens transformed roots indicate that cell suspensions cultures are more promising as potential commercial sources of metabolites such as phenylethanoid glycosides.     

Metal-induced point defects in DNA: model and mechanisms

The aim of this work was to study the role of H3O+ and transition-metal (TM) ions in keto-enol and amino-imino tautomeric transitions in DNA base pairs and depurination. In this regard, we discuss the thermodynamic model of ion-DNA interactions and UV display of double-proton transfer (DPT) in GC. The probabilities and energies of rare tautomeric forms of GC pairs in DNA induced by H3O+ and TMwere determined being in the range from0.02 (forMg2+) to 1 ( forCu2+), and from 0 kcal/m (for Cu2+) to 2.3 kcal/m (for Mg2+), respectively. It was shown that 3'ACC5'/5'TGG3' site of DNA double helix, which corresponds to the only triplet 5'UGG3' of RNA that codes the most valuable amino acid tryptophan, is a good target for TM ions to attack. It was also shown that the only way to obtain the tryptophan-coding 5'UGG3' triplet in RNA via transition-type G --> A point mutation caused by TM ions is their interaction with the site of a DNA double helix, which corresponds to 5'CGG3' triplet of RNA that codes arginine.     

History and evolution of the International Association for Plant Biotechnology (IAPB): 1963–2008

The International Association for Plant Biotechnology (IAPB) was founded in 1963 at the first truly international conference on plant tissue culture, which was organized by Philip R. White. White was a devoted internationalist and was strongly committed to global scientific cooperation. He felt that the time had come for the international tissue culture community to organize so that it could meet regularly and provide a forum to its members for the exchange of ideas and information of mutual interest and use. The various activities of the IAPB since its founding—the publication of its newsletter, its journal, and the proceedings of its quadrennial congresses—faithfully document the remarkable advances in plant biotechnology that were made possible by the successful integration of tissue culture and molecular biology. In particular, the congress proceedings serve as time capsules, providing a wealth of information about the best of science and the most prominent scientists of the time. The history of the IAPB is indeed the history of plant biotechnology.     

UV light-induced DNA damage detection in the unicellular green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Exposure of cells to ultraviolet radiation (UVR) is one of the best studied and most used model system for the examination of the biological effects of DNA damage, its repair and tolerance. The major product after UVR treatment is cyclobutane pyrimidine dimer (TT, TC, CC). Pyrimidine dimers are repaired by a direct reversal called photoreactivation or by excision of damage in a process of nucleotide excision repair. Several methods have been developed for the detection and quantification of pyrimidine dimers in DNA. The technique of Small and Greimann, in which DNA is incubated with the pyrimidine dimer-specific endonuclease, was used for the analysis of mutant strains with impaired excision repair system of the unicellular green alga Chlamydomonas reinhardtii. Another method is based on the binding of specific monoclonal antibodies to pyrimidine dimers. The aim of our work was to compare these two techniques with the use of mutant strains of C. reinhardtii — uvsX1 and uvsX2 which are assumed to be deficient in DNA damage recognition. One of their traits was sensitivity to UVR which could be caused by breakdown of the excision repair pathway. The results suggest that the immuno-approach is suitable for the detection of DNA damage induced by UVR. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Response to oxygen limitation in bacteria of the genus sulfobacillus

For cultures of moderately thermophilic chemolithotrophic bacteria Sulfobacillus sibiricus N1 and SSO, S. thermosulfidooxidans subsp. asporogenes 41, and the thermotolerant strain S. thermotolerans Kr1 grown under forced aeration and in a high medium layer without aeration, growth characteristics, substrate consumption, and exometabolite formation were compared. Sulfobacilli grown under oxygen limitation exhibited greater generation time, longer growth period, cell yield decreased by from 40 to 85% (depending on the strain), suppressed cell respiration ( demonstrated for S. sibiricus N1 ), accumulation of exometabolites (acetate and propionate) in the medium, and emergence of resting forms. For strains N1, SSO, and Kr1, oscillations of Fe(II) and Fe(III) content in the medium were revealed. For S. sibiricus N1 and S. thermotolerans Kr1, grown under hypoxia (0.07% O₂ in the gas phase), coupling of substrate oxidation with Fe(III) reduction was revealed, as well as utilization of Fe(III) as an electron acceptor alternative to oxygen. The role of labile energy and constructive metabolism for survival of sulfobacilli under diverse conditions is discussed.