Chlamydial endocytobionts of free-living amoebae differentially affect the growth rate of their hosts

Bacteria closely related to chlamydiae live and multiply as endocytobionts within free-living amoebae, making these amoebae potential vehicles of new emerging bacterial pathogens of humans. Hartmannella vermiformis containing endobiotic Neochlamydia hartmannellae grew more rapidly than those without endobionts, whilst Acanthamoeba sp. harbouring the Parachlamydia-related endocytobiont UWE25 multiplied more slowly than those without endobionts. The cause for the opposite effect of chlamydial endocytobionts on the growth of their host cells remains unknown.     

Early symbiotic rugosan endobionts in stromatoporoids from the Rhuddanian of Estonia (Baltica)

The earliest known symbiotic rugosan endobionts occur in stromatoporoids from the early Rhuddanian of Estonia. The stromatoporoid Ecclimadictyon nikitini from the Tamsalu Formation contains the rugosan Donacophyllum middendorffii endobiont. A stromatoporoid Clathrodictyon boreale from the Varbola Formation contains Streptelasma estonica and Bodophyllum sp. endobionts. There are up to three endobiotic rugosans per stromatoporoid host. Stromatoporoid hosts were beneficial for symbiotic rugosans as elevated substrates on a seafloor that offered a higher tier for feeding; they also offered enhanced substrate stability. Stromatoporoids of the end‐Ordovician mass‐extinction recovery fauna hosted a diverse fauna of symbiotic endobionts. There were few if any negative effects of this mass extinction on the symbiotic endobionts.     

Evaluation of Bacterial Community Structure and Its Influence on Sulfide Oxidation in a Bio-Leaching Environment

The mechanism of sulfide oxidation by adhering bacteria (direct oxidation mechanism) and by ferric ion in the aqueous phase was studied by quantitative assessment of bacterial activity on the sulfide surface. To probe for the principal bacterial species on the surface and in the supernatant, a library of DNA genes encoding portions of bacterial 16S rRNA was constructed. The PCR-amplified DNA from the bacterial populations was cloned employing PROMEGA's pGEM-T Easy Vector system. The clone frequency indicated that iron-oxidizing bacteria were dominant in the liquid phase, while Acidithiobacillus ferroixdans, which is both sulfur and iron oxidizer was the most prevalent on the sulfide surface. Samples of crystalline pyrite were exposed to the bacterial consortium to evaluate surface alterations caused by bacteria. Chemical (abiotic) oxidation experiments with ferric ion as the oxidant were carried out in parallel with the biological oxidation tests. Changes in the surface topography were monitored by atomic force microscopy (AFM) while changes in surface chemistry were examined by Raman spectroscopy. Bacterial attachment resulted in a 53% increase in the specific surface area in comparison to a 13% increase caused by chemical (ferric ion) oxidation. The oxidation rate was assessed by evaluating the iron release. After corrections for surface area changes, the specific abiotic (oxidation by Fe^{3 +}) and biotic oxidation rates with adhering bacteria were nearly the same (2.6 × 10^{? 9} mol O₂/s/m² versus 3.3 × 10^{? 9} mol O₂/s/m²) at pH = 2 and a temperature of 25°C. The equality of rates implies that the availability of ferric ion as the oxidant is rate limiting.     

Direct evidence for the presence of a rotenone-resistant NADH dehydrogenase on the inner surface of the inner membrane of plant mitochondria

Submitochondrial particles (SMP) were produced from Jerusalem artichoke (Helianthus tuberosus L.) mitochondria by sonication and differential centrifugation. The SMP were about 50% inside-out as measured by the access of reduced cytochrome c to cytochrome c oxidase. Uncoupled NADH oxidation (1 mM NADH) by the SMP was 120 nmol O₂ min^?1mg^?1, which was reduced to 98 nmol O₂ min^?1 (mg mitochondrial protein)^?1 in the presence of EGTA. In contrast, the oxidation of NADH by intact mitochondria was completely inhibited by EGTA (from 182 to 14 nmol O₂ min^?1mg^?1). The EGTA-resistant NADH oxidation by the SMP is ascribed to the NADH dehydrogenase(s) on the inside of the inner membrane and exposed to the medium in the inside-out SMP. In the presence of EGTA it could be shown that two NADH dehydrogenase activities were present in the SMP. One had an apparent K_m of 7 μM for NADH, a V_max of 80 nmol NADH min^?1mg^?1, and was rotenone-sensitive. This dehydrogenase is equivalent to the mammalian Complex I NADH dehydrogenase. The other dehydrogenase, which was rotenone-resistant, had a K_m of 80 μM and a V_max of 131 nmol NADH min^?1mg^?1; it is probably responsible for the rotenone-resistant oxidation of organic acids often observed in plant mitochondria. The redox poise of the pyridine nucleotides had only a small effect on the relative rates of the two internal dehydrogenases. Electron flow through these dehydrogenases appears, therefore, to be regulated mainly by the concentration of NADH in the matrix of the mitochondria.     

Production of Metallogenium-like particles by heterotrophic manganese-oxidizing bacteria collected from a lake

As a model of chemically stratified structure of environment typical to the chemocline of lakes, a semisolid gradient medium was prepared to cultivate heterotrophic manganese-oxidizing bacteria originally collected from a lake. The bacteria growing under the conditions described produced extracellularly Metallogenium-like particles similar to those which are often detected in the chemocline of lakes. This suggested that the naturally occurring Metallogenium-like particles originated in activities of such heterotrophic manganese-oxidizing bacteria. The manganese oxidation activity usually appeared only at the stationary phase of bacterial growth. The oxidation of Mn²⁺ and the formation of Metallogenium-like particles by the bacteria were observed at neutral or slightly acidic pH. not under alkaline conditions, which is a conspicuous difference from the inorganic oxidation of Mn²⁺ by O₂. Bacterial manganese oxidation was stimulated by bicarbonate (50 or 500 M). An experiment of addition of H₂O₂ to the incubation tubes isolated from atmosphere failed to confirm the availability of externally added H₂O₂ as the electron acceptor, suggesting that the bacterial manganese oxidation required the presence of O₂.     

The “one‐point method” for estimating maximum carboxylation capacity of photosynthesis: A cautionary tale

The maximum carboxylation capacity of Rubisco, V_c,max, is an important photosynthetic parameter that is key to accurate estimation of carbon assimilation. The gold‐standard technique for determining V_c,max is to derive V_c,max from the initial slope of an A–C_i curve (the response of photosynthesis, A, to intercellular CO₂ concentration, C_i). Accurate estimates of V_c,max derived from an alternative and rapid “one‐point” measurement of photosynthesis could greatly accelerate data collection and model parameterization. We evaluated the practical application of the one‐point method in six species measured under standard conditions (saturating irradiance and 400 μmol CO₂ mol^?1) and under conditions that would increase the likelihood for successful estimation of V_c,max: (a) ensuring Rubisco‐limited A by measuring at 300 μmol CO₂ mol^?1 and (b) allowing time for acclimation to saturating irradiance prior to measurement. The one‐point method significantly underestimated V_c,max in four of the six species, providing estimates 21%–32% below fitted values. We identified ribulose‐1,5‐bisphosphate‐limited A, light acclimation, and the use of an assumed respiration rate as factors that limited the effective use of the one‐point method to accurately estimate V_c,max. We conclude that the one‐point method requires a species‐specific understanding of its application, is often unsuccessful, and must be used with caution.     

Strain competition restricts colonization of an enteric pathogen and prevents colitis

The microbiota is a major source of protection against intestinal pathogens; however, the specific bacteria and underlying mechanisms involved are not well understood. As a model of this interaction, we sought to determine whether colonization of the murine host with symbiotic non‐toxigenic Bacteroides fragilis could limit acquisition of pathogenic enterotoxigenic B. fragilis. We observed strain‐specific competition with toxigenic B. fragilis, dependent upon type VI secretion, identifying an effector–immunity pair that confers pathogen exclusion. Resistance against host acquisition of a second non‐toxigenic strain was also uncovered, revealing a broader function of type VI secretion systems in determining microbiota composition. The competitive exclusion of enterotoxigenic B. fragilis by a non‐toxigenic strain limited toxin exposure and protected the host against intestinal inflammatory disease. Our studies demonstrate a novel role of type VI secretion systems in colonization resistance against a pathogen. This understanding of bacterial competition may be utilized to define a molecularly targeted probiotic strategy.     

Oxidative Metabolism of 4-Aminobutyrate by Rat Brain Mitochondria: Inhibition by Branched-chain Fatty Acid

Abstract: The oxidation of 4-aminobutyric acid (GABA) by nonsynaptosomal mitochondria isolated from rat forebrain and the inhibition of this metabolism by the branched-chain fatty acids 2-methyl-2-ethyl caproate (MEC) and 2, 2-dimethyl valerate (DMV) were studied. The rate of GABA oxidation, as measured by O₂ uptake, was determined in medium containing either 5 or 100 mM-[K⁺]. The apparent K_m for GABA was 1.16 ± 0.19 mM and the V_max in state 3 was 23.8 ± 5.5 ng-atoms O₂. min^?1. mg protein^?1 in 5 mM-[K⁺]. In a medium with 100 mM-[K⁺] the apparent K_m was 1.11 ± 0.17 mM and V_max was 47.4 ± 5.7 ng-atoms O₂. min^?1. mg protein^?1. The K_m for MEC was determined to be 0.58 ± 0.24 or 0.32 ± 0.08 mM, in 5 or 100 mM-[K⁺], respectively. For DMV, the K_i was 0.28 ± 0.05 or 0.34 ± 0.06 mM, in 5 or 100 mM-[K⁺] medium, respectively. The O₂ uptake of the mitochondria in the presence of GABA was coupled to the formation of glutamate and aspartate; the ratio of oxygen uptake to the rate of amino acid formation was close to the theoretical value of 3. Neither the [K²] nor any of the above inhibitors had any effect on this ratio. The metabolism of exogenous succinic semialdehyde (SSA) by these same mitochondria was also examined. The V_max for utilization of oxygen in the presence of SSA was much greater than that found with exogenously added GABA, indicating that the capacity for GABA oxidation by these mitochondria is not limited by SSA dehydrogenase. In addition, the branched-chain fatty acids did not inhibit the metabolism of exogenously added SSA. Thus, the inhibitors examined apparently act by competitively inhibiting the GABA transaminase system of the mitochondria.     

Kinetics of sulfur oxidation by thiobacillus ferrooxidans

Abstract

Thiobacillus ferrooxidans ATCC 23270 was grown with elemental sulfur as the energy source. Substrate oxidation was measured using a Clark‐type oxygen electrode. Whole cells demonstrated a broad pH optimum for sulfur oxidation between pH 2.0 and 8.0. The V _max and K_sfor sulfur oxidation varied depending on pH. Sulfite was oxidized at 227 nmol O₂/min/mg protein. Thiosulfate oxidation was slow, and tetrathionate oxidation was not detected. At a concentration of 2 mM, sodium azide completely inhibited sulfur, sulfite, and thiosulfate oxidation. Inhibition by N‐ethylmaleimide, antimycin A, and 2‐heptyl‐4‐hydroxyquinoline N‐oxide varied with substrate.     

Scaling with body mass of mitochondrial respiration from the white muscle of three phylogenetically, morphologically and behaviorally disparate teleost fishes

White muscle (WM) fibers in many fishes often increase in size from <50 μm in juveniles to >250 μm in adults. This leads to increases in intracellular diffusion distances that may impact the scaling with body mass of muscle metabolism. We have previously found similar negative scaling of aerobic capacity (mitochondrial volume density, V _mt) and the rate of an aerobic process (post-contractile phosphocreatine recovery) in fish WM. In the present study, we examined the scaling with body mass of oxygen consumption rates of isolated mitochondria (VO_2mt) from WM in three species from different families that vary in morphology and behavior: an active, pelagic species (bluefish, Pomatomus saltatrix), a relatively inactive demersal species (black sea bass, Centropristis striata), and a sedentary, benthic species (southern flounder, Paralichthys lethostigma). In contrast to our prior studies, the measurement of respiration in isolated mitochondria is not influenced by the diffusion of oxygen or metabolites. V _mt was measured in WM and in high-density isolates used for VO_2mt measurements. WM V _mt was significantly higher in the bluefish than in the other two species and VO_2mt was independent of body mass when expressed per milligram protein or per milliliter mitochondria. The size-independence of VO_2mt indicates that differences in WM aerobic function result from variation in V _mt and not to changes in VO_2mt. This is consistent with our prior work that indicated that while diffusion constraints influence mitochondrial distribution, the negative scaling of aerobic processes like post-contractile PCr recovery can largely be attributed to the body size dependence of V _mt.     

Ribosomal DNA shows extremely low genetic divergence in a world-wide distributed,but disjunct and highly adapted marine protozoan (Virgulinella fragilis,Foraminiferida)

Virgulinella fragilis can be mainly observed in different, separated, oxygen-depleted and sulfide-enriched environments around the world and seems to be well adapted to such extreme habitats. Dispersal mechanisms behind this geographical distribution pattern are not yet understood. To analyze the genetic differentiation of geographically isolated populations, we conducted molecular phylogenetic analyses of the small subunit (SSU) ribosomal DNA (rDNA) and internal transcribed spacers (ITS) of rDNA nucleotide sequences in populations of V. fragilis collected in the South Atlantic (upwelling area off Namibia) and in the Pacific (Wellington Harbor, New Zealand, and Namako-ike, Japan). Our molecular analyses revealed SSU rDNA and ITS sequences strikingly similar or identical among these three disjunct populations. Such a low molecular genetic differentiation, a fixation rate converging to zero, could either arise from rapid dispersal, ultraslow mutation rates due to a strictly asexual mode of reproduction, unlimited genetic exchange between populations or the existence of a resting stage for survival under unfavorable conditions. We discuss each explanation and conclude that V. fragilis might possibly represent a protozoan trapped in evolutionary stasis.     

Numbers of iron‐oxidizing bacteria and oxidation potential of sulfur and iron components in coal refuse amended with limestone and sewage sludge

Counts of acidophilic iron‐oxidizing bacteria, ratios of S₂O₃=—S/SO₄=—S and Fe⁺³/Fe⁺², and S₂O₃=—S oxidation potentials were examined over a two‐year period in coal refuse (acid gob) treated with limestone and/or sewage sludge. A non‐amended treatment was used as a control.

No significant difference in population counts of acidophilic iron‐oxidizing bacteria were observed between treatments in either year of the study. S₂O₃=—S/SO₄=—S and Fe⁺³/Fe⁺² ratios indicated active sulfur and iron oxidation suggesting that limestone and/or sewage sludge may be ineffective in suppressing pyrite oxidation. Under optimal conditions, S₂O₃=—S oxidation potentials (in vitro) showed a logarithmic increase in SO₄=—S formation for all four treatments over time. The final pH of the treatments following twenty days of perfusion ranged from 3.06 to 3.59.     

In vivo quantification of neuro‐glial metabolism and glial glutamate concentration using 1H‐[13C] MRS at 14.1T

Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo ¹³C NMR spectroscopy coupled with the infusion labeled glial‐specific substrate, such as acetate. In this study, we infused alpha‐chloralose anesthetized rats with [2‐¹³C]acetate and followed the dynamics of the fractional enrichment (FE) in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using ¹H‐[¹³C] magnetic resonance spectroscopy (MRS) at 14.1T. Applying a two‐compartment mathematical model to the measured time courses yielded a glial tricarboxylic acid (TCA) cycle rate (V_g) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (V_NT) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (V_PC) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (V_x^g and V_xⁿ) were of the same order of magnitude as the respective TCA cycle fluxes. In addition, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations.

     

Cloning and Characterization of the NAD-Dependent 7α-Hydroxysteroid Dehydrogenase from <Emphasis Type="Italic">Bacteroides fragilis</Emphasis>

The NAD-linked 7-hydroxysteroid dehydrogenase (7-HSDH) from Bacteroides fragilis ATCC 25285 was characterized and its gene cloned. The enzyme displayed optimal activities at pH 8.5 (NAD reduction) and 6.5 (NADH oxidation). The lowest K_m and highest V_max values were observed with chenodeoxycholic acid and its conjugates. The protein had subunits of 27.4 kDa and a native size of 110 kDa, suggesting a homotetrameric composition. The enzyme was relatively thermostable, retaining 95% of initial activity after 1 h at 65°C. A DNA probe based on the N-terminal amino acid sequence hybridized to a 2373-bp HindIII fragment of B. fragilis DNA. This fragment was cloned into E. coli and sequenced, revealing a 780-bp open reading frame. The predicted amino acid sequence of the ORF showed strong sequence similarity to three other bacterial 7-HSDHs, all in the short-chain dehydrogenase family. The regulation of expression of this gene is currently under investigation.     

Effect of bedaquiline on the functions of rat liver mitochondria

The paper considers the effects of bedaquiline (BDQ), an antituberculous preparation of the new generation, on rat liver mitochondria. It was shown that 50?μM BDQ inhibited mitochondrial respiration measured with substrates of complexes I and II (glutamate/malate and succinate/rotenone systems respectively) in the states V₃ and V_DNP. At the same time, at concentrations below 50?μM, BDQ slightly stimulated respiration with substrates of complex I in the state V₂. BDQ was also found to suppress, in a dose-dependent manner, the activity of complex II and the total activity of complexes II?+?III of the mitochondrial transport chain. It was discovered that at concentrations up to 10?μM, BDQ inhibited H₂O₂ production in mitochondria. BDQ (10–50?μM) suppressed the opening of Ca²⁺-dependent CsA-sensitive mitochondrial permeability transition pore. The latter was revealed experimentally as the inhibition of Ca²⁺/P_i-dependent swelling of mitochondria, suppression of cytochrome c release, and an increase in the Ca²⁺ capacity of the organelles. BDQ also decreased the rate of mitochondrial energy-dependent K⁺ transport, which was evaluated by the energy-dependent swelling of mitochondria in a K⁺ buffer and DNP-induced K⁺ efflux from the organelles. The possible mechanisms of BDQ effect of rat liver mitochondria are discussed.     

Inhibitory effect of iron‐oxidizing bacteria on ferrous‐promoted chalcopyrite leaching

It is generally accepted that iron‐oxidizing bacteria, Thiobacillus ferrooxidans, enhance chalcopyrite leaching. However, this article details a case of the bacteria suppressing chalcopyrite leaching. Bacterial leaching experiments were performed with sulfuric acid solutions containing 0 or 0.04 mol/dm³ ferrous sulfate. Without ferrous sulfate, the bacteria enhance copper extraction and oxidation of ferrous ions released from chalcopyrite. However, the bacteria suppressed chalcopyrite leaching when ferrous sulfate was added. This is mainly due to the bacterial consumption of ferrous ions which act as a promoter for chalcopyrite oxidation with dissolved oxygen. Coprecipitation of copper ions with jarosite formed by the bacterial ferrous oxidation also causes the bacterial suppression of copper extraction. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 478–483, 1999.     

Extracellular phosphatases in a Mediterranean reservoir: seasonal, spatial and kinetic heterogeneity

1. Epilimnetic alkaline phosphatase activity (APA) was measured in longitudinal profiles of the canyon‐shaped, eutrophic Sau Reservoir (Catalonia, Spain) during the autumn, winter and spring periods of 1997–2000. 2. The spatial pattern of APA depended on lake circulation. During periods of stable stratification, when the ratio of mixed to euphotic depth (z_mix/z_eu < 1.7) was low and the Chlorophyll a (Chl a) concentration high, APA was also comparatively high (0.5–3.4 μmol L^?1 h^?1) and located mostly in the >2‐μm size‐fraction. APA increased towards the dam at the same time as the concentration of soluble reactive phosphorus (SRP) decreased. In periods of unstable stratification, deep mixing (z_mix/z_eu > 2.4) and low Chl a concentration, APA was low (<0.1 μmol L^?1 h^?1) and without longitudinal changes, consistent with a high and stable SRP concentration. 3. A high input of mostly dissolved (in the <0.2‐μm size‐fraction) phosphatases from the river Ter was found in 1997–98. At the river inflow, independently of season and despite a continuously high SRP concentration, APA was approximately 0.7 μmol L^?1 h^?1 and decreased towards the dam within the inflowing, canyon‐like part of the reservoir. 4. Analysis of saturation kinetics revealed the kinetic heterogeneity of APA. Low‐affinity APA was localised in the >2‐μm (algal) size‐fraction while, in the <2‐μm (picoplankton, mainly bacteria + dissolved) size‐fraction, high‐affinity APA, or a mixture of both, was found. The presence of two kinetic components, whose substrate affinities (i.e. in Michaelis constants, K_m) differed, was confirmed statistically in 13 of 18 cases analysed. The range of K_mH values of the high‐affinity component was 0.15–2.4 μmol L^?1, K_mL values of the low‐affinity component ranged from 18 to 275 μmol L^?1.     

Effect of 2,4‐dinitrotoluene on the anaerobic bacterial community in marine sediment

Aims: To study the impact of added 2,4‐dinitrotoluene (DNT) on the anaerobic bacterial community in marine sediment collected from an unexploded ordnance dumping site in Halifax Harbour. Methods and Results: Marine sediment was spiked with 2,4‐DNT and incubated under anaerobic conditions in the presence and absence of lactate. Indigenous bacteria in the sediment removed 2,4‐DNT with subsequent formation of its mono‐ and diamino‐derivatives under both conditions. PCR–DGGE and nucleotide sequencing were used to monitor the change in the bacterial population in sediment caused by the presence of 2,4‐DNT. The results showed that denaturing gradient gel electrophoresis banding patterns of sediment microcosms treated with 2,4‐DNT were different from controls that did not receive 2,4‐DNT. Bacteroidetes, Firmicutes and δ‐Proteobacteria were present in sediment incubated in the absence of 2,4‐DNT. However, several γ‐Proteobacteria became dominant in sediment in the presence of 2,4‐DNT, two of which were 99% similar to Shewanella canadensis and Shewanella sediminis. In the presence of both 2,4‐DNT and lactate, two additional δ‐Proteobacteria were enriched, one closely related (98% similarity) to Desulfofrigus fragile and the other affiliated (96% similarity) to Desulfovibrio sp. In contrast, none of the above four Proteobacteria were enriched in sediment incubated with lactate alone. Conclusions: Presence of 2,4‐DNT led to a significant change in bacterial population of marine sediment with the enrichment of several γ‐ and δ‐Proteobacteria. Significance and Impact of the Study: Our results provided the first evidence on the impact of the pollutant 2,4‐DNT on the indigenous bacterial community in marine sediment, and provided an insight into the composition of bacterial community that degrade 2,4‐DNT.     

Ellagic acid mitigates arsenic‐trioxide‐induced mitochondrial dysfunction and cytotoxicity in SH‐SY5Y cells

In the current study, neuroprotective significance of ellagic acid (EA, a polyohenol) was explored by primarily studying its antioxidant and antiapoptotic potential against arsenic trioxide (As₂O₃)‐induced toxicity in SH‐SY5Y human neuroblastoma cell lines. The mitigatory effects of EA with particular reference to cell viability and cytotoxicity, the generation of reactive oxygen species, DNA damage, and mitochondrial dynamics were studied. Pretreatment of SH‐SY5Y cells with EA (10 and 20 μM) for 60 min followed by exposure to 2 μM As₂O₃ protected the SH‐SY5Y cells against the harmful effects of the second. Also, EA pre‐treated groups expressed improved viability, repaired DNA, reduced free radical generation, and maintained altered mitochondrial membrane potential than those exposed to As₂O₃ alone. EA supplementation also inhibited As₂O₃‐induced cytochrome c expression that is an important hallmark for determining mitochondrial dynamics. Thus, the current investigations are more convinced for EA as a promising candidate in modulating As₂O₃‐induced mitochondria‐mediated neuronal toxicity under in vitro system.