Extracytoplasmic processes impaired by inactivation of trxA (thioredoxin gene) in Bacillus subtilis

The trxA gene is regarded as essential in Bacillus subtilis, but the roles of the TrxA protein in this gram-positive bacterium are largely unknown. Inactivation of trxA results in deoxyribonucleoside and cysteine or methionine auxotrophy. This phenotype is expected if the TrxA protein is important for the activity of the class Ib ribonucleotide reductase and adenosine-5'-phosphosulfate/3'-phosphoadenosine-5'-phosphosulfate reductase. We demonstrate here that a TrxA deficiency in addition causes defects in endospore and cytochrome c synthesis. These effects were suppressed by BdbD deficiency, indicating that TrxA in the cytoplasm is the primary electron donor to several different thiol-disulfide oxidoreductases active on the outer side of the B. subtilis cytoplasmic membrane.     

Exchange of CO2, CH4 and N2O between the atmosphere and two northern boreal ponds with catchments dominated by peatlands or forests

Concentrations of carbon dioxide (CO₂), methane (CH₄) and nitrous oxide (N₂O) in the water column and their exchange at the water/air interface were studied during the open water period in two freshwater ponds with different catchment characteristics in the northern boreal zone in Finland; either peatlands or coniferous upland forests dominated the catchment of the ponds. Both ponds were supersaturated with dissolved CO₂ and CH₄ with respect to the equilibrium with the atmosphere, but were close to the equilibrium with N₂O. The mean CO₂ efflux from the pond was higher in the peatland-dominated catchment (22 mg m^–2 h^–1) than in the forested catchment (0.7 mg m^–2 h^–1), whereas the mean CH₄ emissions were similar (7.6 and 3.5 mg m^–2 d^–1, respectively). The fluxes of N₂O were generally negligible. The higher CO₂ concentrations and efflux in the pond with the peatland-dominated catchment were attributed to a greater input of allochthonous carbon to that pond from its catchment due to its higher water colour and higher total organic carbon (TOC) concentration. The water pH, which also differed between the ponds, could additionally affect the CO₂ dynamics. Since the catchment characteristics can regulate aquatic carbon cycles, catchment-scale studies are needed to attain a deeper understanding of the aquatic greenhouse gas dynamics.     

Quantitative Analysis of Cereulide, the Emetic Toxin of Bacillus cereus, Produced under Various Conditions

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 μg ml⁻¹. The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 μg of cereulide g⁻¹ (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 μg of cereulide ml⁻¹ in liquid medium at room temperature (21 ± 1°C) in 1 to 3 days, during the stationary growth phase when cell density was 2 × 10⁸ to 6 × 10⁸ CFU ml⁻¹. Cereulide production at temperatures at and below 8°C or at 40°C was minimal.     

Effects of polarization in the presence and absence of biocides on biofilms in a simulated paper machine water

The antifouling potential of electric polarization combined and not combined with biocides was studied in nonsaline warm water with high organic content. Deinococcus geothermalis is a bacterium known for forming colored biofilms in paper machines and for its persistence against cleaning and chemical treatments. When D. geothermalis biofilms grown for 24 h in simulated paper machine water were exposed to cathodic or cathodically weighted pulsed polarization at least 60% (P < 0.05) of the biofilms were removed from stainless steel (AISI 316L). Biofilm removal by 25 ppm (effective substances 5-25 ppm) of oxidizing biocides (bromochloro-5,5-dimethylhydantoin, 2,2-dibromo-2-cyanoacetamide, peracetic acid) increased to 70% when combined with cathodically weighted pulsed polarization. Using a novel instrument that allows real-time detection of reactive oxygen species (ROS) we showed that the polarization program effective in antifouling generated ROS in a pulsed manner on the steel surface. We thus suggest that the observed added value of oxidative biocides combined with polarization depended on ROS. This suggestion was supported by the finding that a reductive biocide, methylene bisthiocyanate, counteracted the antifouling effect of polarization.     

Degradation of paper mill water components in laboratory tests with pure cultures of bacteria

The degradation of dissolved and colloidal substances from thermomechanical pulp (TMP) by bacteria isolated from a paper mill was studied in a laboratory slide culture system.Burkholderia cepacia strains hydrolysed triglycerides to free fatty acids, and the liberated unsaturated fatty acids were then degraded to some extent. Saturated fatty acids were not notably degraded. However, the branched anteiso-heptadecanoic fatty acid was degraded almost like the unsaturated fatty acids. About 30% of the steryl esters were degraded during 11 days, increasing the concentrations of free sterols. Approximately 25% of the dehydroabietic, and 45% of the abietic and isopimaric resin acids were degraded during 11 days. The degree of unsaturation seemed to be of greater importance for the degradation of fatty acids than the molar mass. No degradation of dissolved hemicelluloses could be observed with any of the nine bacterial strains studied. Burkholderia cepacia strains and one Bacillus coagulans strain degraded monomeric fructose and glucose in winter TMP water, but in summer TMP water, with much lower sugar concentrations, also otherBacillus strains degraded monomeric sugars.     

Contact-dependent inhibition of angiogenesis by cardiac fibroblasts in three-dimensional fibrin gels in vitro: implications for microvascular network remodeling and coronary collateral formation

Angiogenesis and coronary artery collateral formation can improve blood flow and thereby prevent myocardial ischemia. The role of perivascular fibroblasts in neovascularization remains incompletely understood. Here we investigated the effects of epicardial and myocardial fibroblasts on angiogenesis in vitro by using a serum-free microcarrier-based fibrin gel angiogenesis system. To clearly distinguish between different cell types, we either stained endothelial cells or fibroblasts in the living with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine-perchlorate (DiI). In cocultures, low numbers of heart fibroblasts stimulated endothelial sprouting, and capillary growth was also induced by fibroblast-conditioned media, indicating a paracrine mechanism. Capillary formation was decreased by increasing the density of fibroblasts in the cocultures, indicating contact-dependent inhibition. Using time-lapse studies, it turned out that close contacts between fibroblasts and endothelial cells resulted in rapid retraction of endothelial cells or, rarely, in cell death. Depending on the local ratio of fibroblasts to endothelial cell numbers, fibroblasts determined the location of capillary growth and the size of developing capillaries and thereby contributed to capillary network remodeling. In contrast to primary heart fibroblasts, NIH 3T3 fibroblasts did not display contact-dependent inhibition of endothelial sprouts. NIH fibroblasts were frequently seen in close association with endothelial capillaries, resembling pericytes. Contact-dependent inhibition of angiogenesis by epicardial fibroblasts could not be reversed by addition of neutralizing anti-TGF-β1 antibodies, by addition of serum, of medium conditioned by hypoxic tumor cells or myocardium, by various cytokines or by growing cocultures under hypoxic conditions. Our results implicate a pivotal role of periendothelial mesenchymal cells for the regulation of microvascular network remodeling and collateral formation. Received: 15 September 1997 / Accepted: 6 April 1998     

In vitro studies on intestinal calcium and phosphate transport in horses

Transepithelial transport mechanisms play a key role in regulating the absorption and secretion of calcium (Ca^2 +) and inorganic phosphate (P_i) in the gastrointestinal tract. Although intestinal disorders with imbalances in macromineral homeostasis are frequently observed in horses, available data on intestinal Ca^2 + and P_i transport are limited. The aim of the present study was to characterize the intestinal Ca^2 + and P_i transport functionally by using the in vitro radioisotope tracer technique with Ussing chambers and to identify components involved in Ca^2 + transport at both mRNA and protein level. Among the different intestinal segments, the duodenum showed significant and highest active Ca^2 + absorption. The findings from RT-PCR and Western blot analysis suggest that the epithelial Ca^2 + channel TRPV6, the cytosolic calcium binding protein calbindin-D_9K and the plasma membrane calcium ATPase PMCA may be involved in active transcellular Ca^2 + transport. Regarding the P_i transport, the results indicate significant active P_i secretion in the jejunum, but the contributing mechanisms remain unclear. A significant inhibiting effect of ouabain as an antagonist of the basolateral Na⁺/K⁺-ATPase on the serosal-to-mucosal P_i transport suggests a pivotal role of Na⁺ in jejunal P_i transport in the horse.     

Improving Biodegradation of VOCs in Soil by Controlling Volatilization

The use of microbial inoculum and a hydrocarbon adsorbent as a soil amendment was examined to improve bioremediation efficacy of soil contaminated by volatile hydrocarbons. Biodegradation and volatilization losses of VOCs were assessed under contained composting in the laboratory and technical scales. Rhodococcus opacus GM-14, a degrader of a multitude of different hydrocarbons was used as an inoculum and activated carbon as a VOC adsorbent on a laboratory scale. Inoculating soil with R. opacus (0.02 mg R. opacus biomass per 1 mg of benzene) reduced volatilization of benzene from 80% to 40%. Amending the soil with activated carbon reduced volatilization of benzene to 15% and further to 4% when used together with R. opacus. Both amendments promoted mineralization when used separately but slowed down the mineralization when combined. Activated carbon improved the biodegradation of VOCs also during technical scale compostings (700-1100 kg of soil with 1.6-2.4 kg of VOC) from 30-40% to 86% and reduced volatilization from 40-50% to 2-5%. Reduction of VOC volatilization by use of the activated carbon improved the efficiency of VOC biodegradation on a technical scale. The activated carbon addition improves the occupational safety at the contaminated site and during transport.     

Antibodies to a full-length VAR2CSA immunogen are broadly strain-transcendent but do not cross-inhibit different placental-type parasite isolates

The high molecular weight, multidomain VAR2CSA protein mediating adhesion of Plasmodium falciparum-infected erythrocytes in the placenta is the leading candidate for a pregnancy malaria vaccine. However, it has been difficult so far to generate strong and consistent adhesion blocking antibody responses against most single-domain VAR2CSA immunogens. Recent advances in expression of the full-length recombinant protein showed it binds with much greater specificity and affinity to chondroitin sulphate A (CSA) than individual VAR2CSA domains. This raises the possibility that a specific CSA binding pocket(s) is formed in the full length antigen and could be an important target for vaccine development. In this study, we compared the immunogenicity of a full-length VAR2CSA recombinant protein containing all six Duffy binding-like (DBL) domains to that of a three-domain construct (DBL4-6) in mice and rabbits. Animals immunized with either immunogen acquired antibodies reacting with several VAR2CSA individual domains by ELISA, but antibody responses against the highly conserved DBL4 domain were weaker in animals immunized with full-length DBL1-6 recombinant protein compared to DBL4-6 recombinant protein. Both immunogens induced cross-reactive antibodies to several heterologous CSA-binding parasite lines expressing different VAR2CSA orthologues. However, antibodies that inhibited adhesion of parasites to CSA were only elicited in rabbits immunized with full-length immunogen and inhibition was restricted to the homologous CSA-binding parasite. These findings demonstrate that partial and full-length VAR2CSA immunogens induce cross-reactive antibodies, but inhibitory antibody responses to full-length immunogen were highly allele-specific and variable between animal species.     

Genes coding for cytochromec oxidase inParacoccus denitrificans

Several loci on theParacoccus denitrificans chromosome are involved in the synthesis of cytochromec oxidase. So far three genetic loci have been isolated. One of them contains the structural genes of subunits II and III, as well as two regulatory genes which probably code for oxidase-specific assembly factors. In addition, two distinct genes for subunit I have been cloned, one of which is located adjacent to the cytochromec ₅₅₀ gene. An alignment of six promoter regions reveals only short common sequences.