Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

Growth of an anaerobic sulfate-reducing bacterium sustained by oxygen respiratory energy conservation after O2-driven experimental evolution

Desulfovibrio species are representatives of microorganisms at the boundary between anaerobic and aerobic lifestyles, since they contain the enzymatic systems required for both sulfate and oxygen reduction. However, the latter has been shown to be solely a protective mechanism. By implementing the oxygen-driven experimental evolution of Desulfovibrio vulgaris Hildenborough, we have obtained strains that have evolved to grow with energy derived from oxidative phosphorylation linked to oxygen reduction. We show that a few mutations are sufficient for the emergence of this phenotype and reveal two routes of evolution primarily involving either inactivation or overexpression of the gene encoding heterodisulfide reductase. We propose that the oxygen respiration for energy conservation that sustains the growth of the O₂-evolved strains is associated with a rearrangement of metabolite fluxes, especially NAD⁺/NADH, leading to an optimized O₂ reduction. These evolved strains are the first sulfate-reducing bacteria that exhibit a demonstrated oxygen respiratory process that enables growth.     

Deletion of PsbM in tobacco alters the QB site properties and the electron flow within photosystem II

Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.     

Recent emergence of serogroup C meningococcal disease in Greece

The number of cases of meningococcal disease reported to the Meningitis Reference Laboratory in Athens rose dramatically in 1996-1997. The aims were (1) to determine if the increase was due to introduction of new strains, (2) to assess the geographic and age distribution of the cases, (3) to compare antibiotic sensitivity patterns of the current isolates with strains from the early 1990s. In 1993-1994, 15/19 (74%) of the cases for which information on age was available were in children < or = 5 years; in 1995-1997, 80/179 (45%) of cases were in children < or = 5 years and 99 (55%) in the older age range (P < 0.02). From 593 cases in 1993 1997, 214 (36%) isolates were available for characterisation. Serogroup B was predominant in the early 1990s, but by 1997, serogroup C accounted for 46/72 (64%) of isolates and serogroup B for 25/72 (35%). Serogroup B was predominant in children < or = 5 years (44/78, 56%) but only 19/99 (18%) of older children and adults (P=0.0000005). Sulfonamide resistance decreased from 10/22 (45%) in 1993-1994 to 27/192 (14%) in 1995-1997 (P<0.01). Multilocus enzyme electrophoresis of 70 strains obtained during this period identified the epidemic ET-15 clone in 24 (34.3)%. The profiles of the Greek ET-15 isolates were identical to C:2a:P1.2(P1.5) strains responsible for the epidemic in the Czech Republic which began in 1993. This genotype was not found in Greek strains isolated prior to 1993. We conclude that the increase in meningococcal disease is due to introduction of the epidemic serogroup C:2a:P1.2(P1.5) strain responsible for disease in the Czech Republic and Canada.     

mau-2 acts cell-autonomously to guide axonal migrations in Caenorhabditis elegans

The gene mau-2 has been found to be required for the guidance of cellular and axonal migrations along both the anteroposterior and the dorsoventral body axes during the development of the nematode C. elegans. We show that mau-2 encodes a novel, previously uncharacterized protein that is highly conserved among animals. Maternal mau-2 gene expression is sufficient for normal development until the fourth larval stage, and a MAU-2::GFP fusion protein localizes to the cytoplasm of neurones. mau-2 is ubiquitously expressed in embryos by late gastrulation and becomes predominantly expressed in the nervous system as morphogenesis progresses. Expression of mau-2 within individual neurones rescues the guidance defects of mau-2 mutants, indicating that mau-2 functions cell-autonomously. Altering the activity of both the dorsal repellent slt-1 and mau-2 leads to the abnormal dorsal projection of the AVM axon, a phenotype that is novel and specific to the interaction of these two genes, indicating that mau-2 participates in the guidance of AVM by a slt-1-independent mechanism. Taken together, mau-2 defines a novel guidance factor that might be involved in the intracellular processing of guidance cues encountered by migrating cells and axons during development.     

Dietary monounsaturated versus polyunsaturated fatty acids: which is really better for protection from coronary heart disease?

PURPOSE OF REVIEW: The purpose is to evaluate recent findings concerning dietary fats and the risk of coronary heart disease. Monounsaturated fatty acids are often regarded as healthy, and many have recommended their consumption instead of saturated fatty acids and polyunsaturated fatty acids. Support for the benefits of monounsaturated fatty acids comes largely from epidemiological data, but they have not been an isolated, single variable in such studies. Beneficial effects on the plasma lipid profile and LDL oxidation rates have also been identified. More recent findings have questioned the impact of suspected beneficial effects on coronary heart disease, indicating that studies with more conclusive endpoints are needed. RECENT FINDINGS: Human dietary studies often produce conflicting results regarding the effects of monounsaturated and polyunsaturated fatty acids on the plasma lipid profile. Monounsaturated and polyunsaturated fatty acids both appear to reduce total and LDL-cholesterol compared with saturated fatty acids; however, the effect on HDL is less clear. Lowered HDL levels in response to low-fat or polyunsaturated fatty acid diets and the decreased protection from oxidation of polyunsaturated fatty acid-enriched LDL may not indicate increased coronary heart disease risk. Several lines of evidence also suggest that polyunsaturated fatty acids may protect against atherosclerosis. SUMMARY: Recommendations to substitute monounsaturated fatty acids for polyunsaturated fatty acids or a low-fat carbohydrate diet seem premature without more research into the effects on the development of atherosclerosis. Current opinions favoring monounsaturated fatty acids are based on epidemiological data and risk factor analysis, but are questioned by the demonstrated detrimental effects on atherosclerosis in animal models.     

Phytoextraction of Risk Elements by Willow and Poplar Trees

To characterize the phytoextraction efficiency of two clones of willow trees (Salix x smithiana Willd., Salix rubens) and two clones of poplar trees (Populus nigra x maximowiczii, Populus nigra Wolterson) were planted in contaminated soil (0.4–2.0 mg Cd.kg^?1, 78–313 mg Zn.kg^?1, 21.3–118 mg Cu.kg^?1). Field experiment was carried out in Czech Republic. The study investigated their ability to accumulate heavy metals (Cd, Zn, and Cu) in harvestable plant parts. The poplars produced higher amount of biomass than willows. Both Salix clones accumulated higher amount of Cd, Zn and Cu in their biomass (maximum 6.8 mg Cd.kg^?1, 909 mg Zn.kg^?1, and 17.7 mg Cu.kg^?1) compared to Populus clones (maximum 2.06 mg Cd.kg^?1, 463 mg Zn.kg^?1, and 11.8 mg Cu.kg^?1). There were no significant differences between clones of individual species. BCs for Cd and Zn were greater than 1 (the highest in willow leaves). BCs values of Cu were very low. These results indicate that Salix is more suitable plant for phytoextraction of Cd and Zn than Populus. The Cu phytoextraction potential of Salix and Populus trees was not confirmed in this experiment due to low soil availability of this element.     

Ethylene triggers needle abscission in root-detached balsam fir

Post-harvest needle abscission is a major challenge for Christmas tree and greenery industries. It was hypothesized that ethylene triggers abscission in balsam fir. Three experiments were conducted to test this hypothesis. In experiment 1, 70 balsam fir branches were collected, placed in water, and ethylene evolution was observed over time. In experiment 2, a 2 × 5 factorial experiment was designed to determine the effect of exogenous ethylene and an ethylene receptor blocker, 1-methylcyclopropene (1-MCP), on needle abscission. In experiment 3, a 2 × 6 factorial experiment was designed to determine the effect of exogenous ethylene and an ethylene inhibitor, aminoethoxyvinylglycine (AVG), on needle abscission. It was found that ethylene evolution was the highest 1–2 days prior to needle abscission, which was consistent in untreated branches and branches exposed to exogenous ethylene. Exposure to exogenous ethylene significantly decreased needle retention by 63%. When ethylene receptors were blocked by 1-MCP, needle retention increased by 147% despite the presence of ethylene and increased by 73% in the absence of ethylene when compared to the respective controls. When endogenous ethylene synthesis was inhibited by AVG, there was no improvement in needle retention in the presence of ethylene, but there was a 113% increase in needle retention in the absence of exogenous ethylene. Ethylene is strongly implicated as the signal triggering abscission in root-detached balsam fir.     

Characteristics of Gloeophyllum trabeum alcohol oxidase, an extracellular source of H2O2 in brown rot decay of wood

A novel alcohol oxidase (AOX) has been purified from mycelial pellets of the wood-degrading basidiomycete Gloeophyllum trabeum and characterized as a homooctameric nonglycosylated protein with native and subunit molecular masses of 628 and 72.4 kDa, containing noncovalently bonded flavin adenine dinucleotide. The isolated AOX cDNA contained an open reading frame of 1,953 bp translating into a polypeptide of 651 amino acids displaying 51 to 53% identity with other published fungal AOX amino acid sequences. The enzyme catalyzed the oxidation of short-chain primary aliphatic alcohols with a preference for methanol (K(m) = 2.3 mM, k(cat) = 15.6 s(-1)). Using polyclonal antibodies and immunofluorescence staining, AOX was localized on liquid culture hyphae and extracellular slime in sections from degraded wood and on cotton fibers. Transmission electron microscopy immunogold labeling localized the enzyme in the hyphal periplasmic space and wall and on extracellular tripartite membranes and slime, while there was no labeling of hyphal peroxisomes. AOX was further shown to be associated with membranous or slime structures secreted by hyphae in wood fiber lumina and within the secondary cell walls of degraded wood fibers. The differences in AOX targeting compared to the known yeast peroxisomal localization were traced to a unique C-terminal sequence of the G. trabeum oxidase, which is apparently responsible for the protein's different translocation. The extracellular distribution and the enzyme's abundance and preference for methanol, potentially available from the demethylation of lignin, all point to a possible role for AOX as a major source of H(2)O(2), a component of Fenton's reagent implicated in the generally accepted mechanisms for brown rot through the production of highly destructive hydroxyl radicals.