David Lloyd

Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide

The flavohaemoglobin Hmp of Escherichia coli is inducible by nitric oxide (NO) and provides protection both aerobically and anaerobically from inhibition of growth by NO and agents that cause nitrosative stress. Here we report rapid kinetic studies of NO binding to Fe(III) Hmp with a second order rate constant of 7.5×105 M−1 s−1 to generate a nitrosyl adduct that was stable anoxically but decayed in the presence of air to reform the Fe(III) protein. NO displaced CO bound to dithionite‐reduced Hmp but, remarkably, CO recombined after only 2 s at room temperature indicative of NO reduction and dissociation from the haem. Addition of NO to anoxic NADH‐reduced Hmp also generated a nitrosyl species which persisted while NADH was oxidised. These results are consistent with direct demonstration by membrane‐inlet mass spectrometry of NO consumption and nitrous oxide production during anoxic incubation of NADH‐reduced Hmp. The results demonstrate a new mechanism by which Hmp may eliminate NO under anoxic growth conditions.

Purification and characterization of hydroxycinnamate decarboxylase from Brettanomyces anomalus

Abstract The yeast, Brettanomyces anomalus , produces a hydroxycinnamic acid decarboxylase which is active toward ferulic acid, p -coumaric acid, and caffeic acid. The enzyme transforms these hydroxycinnamic acids to hydroxystyrenes by the removal of the carboxyl group from the C3 side chain. We have purified this enzyme 235-fold from B. anomalus NCYC 615 using Mono Q ion exchange, Phenyl Superose, and Superose 12 column chromatography. Enzyme activity was found to be optimal at 40°C and pH 6.0 and was enhanced by EDTA, Mg 2+ , and Cr 3+ . Fe 3+ , Ag + , and SDS completely inhibited the activity. Kinetic studies indicated a K m of 1.15 m m and a V max of 13,494 nmol min −1 mg −1 for ferulic acid and a K m of 1.55 mM and a V max of 22,256 nmol min −1 mg −1 for p -coumaric acid. Using gel filtration, an apparent molecular mass of 39.8 kDa was estimated. The decarboxylase was inactive toward both o - and m -coumaric acid and toward cinnamic acid, indicating that the para -hydroxy group is essential for activity.

Aerobic denitrification in soils and sediments: From fallacies to factx

Denitrification is the key step of the nitrogen cycle in which gaseous end products are released from the nitrate of terrestrial and aquatic environments. Although this process has always been regarded as an anaerobic one, recent research indicates that aerobic denitrification can be demonstrated with laboratory cultures and suggests that it may be widespread environmentally. Thus, denitrifying bacteria are both taxonomically and physiologically diverse, and may be predominantly aerobic. Simultaneous use of O(2) and NO(3)(-) as alternative terminal oxidants is not precluded in many bacteria, although the use of NO(3)(-) when O(2) is available is of no known advantage.

Rapid estimation of bacterial antibiotic susceptibility with flow cytometry

Bacterial antibiotic susceptibility was rapidly estimated for Escherichia coli and Staphylococcus spp. by flow cytometry. This was achieved by measuring the uptake of a negatively charged membrane potential sensitive dye bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol and observing changes in low‐angle light scatter (excitation light scattered by up to 15°). Estimations of ampicillin, gentamicin and ciprofloxacin susceptibilities were possible within 2–5 h from a plate culture, depending on the species and antibiotic used. This includes the time necessary to establish steady‐state growth in liquid culture.

THE OCCURRENCE AND FUNCTIONS OF ULTRADIAN RHYTHMS

Ultradian oscillations with periods between 5 min and 4 h have been described in cell‐free extracts, single‐celled eukaryotes, cultured cells and embryos. Whereas some of these potentially oscillatory systems (e.g. glycolysis) may only exhibit this type of behaviour rarely if at all in vivo, other ultradian oscillators in lower eukaryotes are rhythms and probably have timekeeping functions. Rhythms with ultradian periods of 10 min to 20 h in oxygen consumption and carbon dioxide production have also been studied in endotherm animals: these rhythms may be modified by variations of environmental parameters and by circadian and infradian synchronizers. Interspecies and interstrain differences strongly suggest that these rhythms are endogenous and have a genetic origin. We suggest that the temporal organization of biochemical and physiological processes facilitates optimization of thermodynamic maintenance of the organism within the random fluctuations of its physicochemical environment and contributes to genetic selection.

Ferredoxin-dependent reduction of nitroimidazole derivatives in drug-resistant and susceptible strains of Trichomonas vaginalis.

The inhibitory effect of a range of nitroimidazole-derivatives on H2 production by metronidazole resistant (CDC-85) and susceptible (C1-NIH) Trichomonas vaginalis strains was investigated. The 2-, 4-, and 5-nitro-derivatives used had one-electron reduction potentials within the range -250 to -525 mV. Nitroimidazole concentrations giving 50% inhibition of H2 production (kiH2) for compounds with one-electron reduction potentials in the range -250 to -425 mV were found to be similar for both strains tested. Compounds with one-electron reduction potentials below -425 mV give 10-fold higher KiH2 values for the metronidazole resistant isolate. Both strains showed increased KiH2 for compounds with potentials lower than -500 mV. The addition of 2.1 kPa (0.02 atm) O2 to the gas phase resulted in increasing the kiH2 values for all the compounds tested, but had the greater effect on results obtained with the resistant isolate using nitroimidazoles in the range -425 to -490 mV. The results enable the proposal that the resistant isolate CDC-85 has a ferredoxin with altered redox properties or reduced intracellular levels.

Giardia lamblia produces alanine anaerobically but not in the presence of oxygen.

Proton nuclear magnetic resonance was used to follow glucose metabolism in Giardia lamblia. Under strictly anaerobic conditions this organism produces equimolar ethanol and alanine as well as CO2 and some acetate. Aerobically the production of both alanine and ethanol are inhibited and more acetate and CO2 are formed. These changes in the balance of products are reversible over the range 0-46 microM O2. In the presence of 46 microM O2, alanine was not detectable. The O2-sensitivity of alanine production may highlight the necessity for redox-balancing reactions in an organism exposed in situ to fluctuating concentrations of O2.

Trichomonas vaginalis requires traces of oxygen and high concentrations of carbon dioxide for optimal growth.

The effects of O2 and CO2 on the growth in culture of Trichomonas vaginalis strain C1-NIH were investigated. Growth under pre-purified N2 in the absence of CO2 supplementation gave a doubling time of 4.4 h; when traces of O2 (less than 0.25 microM) were present, the doubling time was 3.5 h. Organisms grew most rapidly (doubling time 2.3 h) with traces of O2 (less than 0.25 microM) and with the CO2 level controlled at 5 mM. The balance of fermentation products from maltose was greatly influenced by supplied gases. Under strictly anaerobic conditions at 5 mM CO2, equimolar glycerol and lactate accounted for more than 95% of the measured products, whereas lower CO2 increased acetate production. Under microaerobic conditions (O2 less than 0.25 microM) acetate was the major product when CO2 was limited to that evolved endogenously; again 5 mM CO2 favoured glycerol and lactate production. Activities of key enzymes measured in cell-free extracts (pyruvate:ferredoxin oxidoreductase, hydrogenase, glycerol kinase, malate dehydrogenase (decarboxylating) and lactate dehydrogenase) altered with growth conditions commensurately with observed changes in metabolic flux patterns. These results suggest that T. vaginalis is optimally adapted to conditions it experiences in situ in the vagina (traces of O2, high CO2).

A membrane-inlet mass spectrometer miniprobe for the direct simultaneous measurement of multiple gas species with spatial resolution of 1 mm

Abstract The design and construction of a membrane-covered gas miniprobe inlet using a quadrupole mass spectrometer is described. The probe is a metre-long steel capillary tube (o.d. 1.6 mm) terminating in a 4 cm 300 μm o.d. hypodermic needle. Response times for H 2 , CH 4 , N 2 , O 2 and CO 2 in gaseous or liquid phases are of the order of minutes ( t 100% ). Gas concentrations of the order of 1 μM can be measured with a spatial resolution of the order of 1 mm. Examples are given of the vertical distributions of O 2 and CO 2 in a freshwater sediment, and of O 2 , CO 2 , and CH 4 in a peat core. The characteristics of this system are discussed in detail in terms of the usefulness of the device for localized ecological gas measurements.

Hypothesis: the central oscillator of the circadian clock is a controlled chaotic attractor

Controlled chaos may be important for the generation of rhythmic behaviour in living systems. A model is proposed in which the central circadian oscillator is a chaotic attractor. Whereas a limit cycle mechanism (previously invoked to explain circadian clocks as well as ultradian clocks and cell division cycles) can provide only a single stable periodic orbit, a chaotic attractor can generate rich dynamic behaviour. Control by feedback makes accessible a selected stabilized orbit; this can be chosen so as to optimize system performance. Such a system can accommodate a wide variety of requirements, e.g. that a single clock mutation can affect both period and temperature compensation, and the generation of higher periods from an ultradian oscillator. Simultaneous operation of more than one clock (with differing periods) may require a high-dimension chaotic attractor. Attractive features of such a model include versatility of period selection (e.g. as in the per mutants of Drosophila) and the use of control elements of the type already well known in metabolic circuitry.