Denitrifying Pseudomonas aeruginosa: some parameters of growth and active transport.
Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity. (+info)
Ferritin mutants of Escherichia coli are iron deficient and growth impaired, and fur mutants are iron deficient.
Escherichia coli contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes (ftnA and bfr) were inactivated by replacing the chromosomal ftnA and bfr genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants (ftnA::spc and bfr::kan) and a double mutant (ftnA::spc bfr::kan) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the bfr mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the ftnA and ftnA bfr mutants remained unchanged. The ftnA and ftnA bfr mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation (fur) mutants also had very low iron contents (2.5-fold less iron than Fur+ strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the ftnA and fur mutants were confirmed by Mossbauer spectroscopy, which further showed that the low iron contents of ftnA mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the fur mutation, ftnA and bfr mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of "reactive ferrous iron." It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling the expression of iron-containing proteins. The role of Bfr remains unclear. (+info)
Molecular characterization of the nitrite-reducing system of Staphylococcus carnosus.
Characterization of a nitrite reductase-negative Staphylococcus carnosus Tn917 mutant led to the identification of the nir operon, which encodes NirBD, the dissimilatory NADH-dependent nitrite reductase; SirA, the putative oxidase and chelatase, and SirB, the uroporphyrinogen III methylase, both of which are necessary for biosynthesis of the siroheme prosthetic group; and NirR, which revealed no convincing similarity to proteins with known functions. We suggest that NirR is essential for nir promoter activity. In the absence of NirR, a weak promoter upstream of sirA seems to drive transcription of sirA, nirB, nirD, and sirB in the stationary-growth phase. In primer extension experiments one predominant and several weaker transcription start sites were identified in the nir promoter region. Northern blot analyses indicated that anaerobiosis and nitrite are induction factors of the nir operon: cells grown aerobically with nitrite revealed small amounts of full-length transcript whereas cells grown anaerobically with or without nitrite showed large amounts of full-length transcript. Although a transcript is detectable, no nitrite reduction occurs in cells grown aerobically with nitrite, indicating an additional oxygen-controlled step at the level of translation, enzyme folding, assembly, or insertion of prosthetic groups. The nitrite-reducing activity expressed during anaerobiosis is switched off reversibly when the oxygen tension increases, most likely due to competition for electrons with the aerobic respiratory chain. Another gene, nirC, is located upstream of the nir operon. nirC encodes a putative integral membrane-spanning protein of unknown function. A nirC mutant showed no distinct phenotype. (+info)
Combination of fluorescent in situ hybridization and microautoradiography-a new tool for structure-function analyses in microbial ecology.
A new microscopic method for simultaneously determining in situ the identities, activities, and specific substrate uptake profiles of individual bacterial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA-targeted oligonucleotide probes and microautoradiography. This method was evaluated by using defined artificial mixtures of Escherichia coli and Herpetosiphon aurantiacus under aerobic incubation conditions with added [3H]glucose. Subsequently, we were able to demonstrate the potential of this method by visualizing the uptake of organic and inorganic radiolabeled substrates ([14C]acetate, [14C]butyrate, [14C]bicarbonate, and 33Pi) in probe-defined populations from complex activated sludge microbial communities by using aerobic incubation conditions and anaerobic incubation conditions (with and without nitrate). For both defined cell mixtures and activated sludge, the method proved to be useful for simultaneous identification and analysis of the uptake of labeled substrates under the different experimental conditions used. Optimal results were obtained when fluorescently labeled oligonucleotides were applied prior to the microautoradiographic developing procedure. For single-cell resolution of FISH and microautoradiographic signals within activated sludge flocs, cryosectioned sample material was examined with a confocal laser scanning microscope. The combination of in situ rRNA hybridization techniques, cryosectioning, microautoradiography, and confocal laser scanning microscopy provides a unique opportunity for obtaining cultivation-independent insights into the structure and function of bacterial communities. (+info)
Influences of low intensity exercise on body composition, food intake and aerobic power of sedentary young females.
The present study was designed to investigate the influences of aerobic training on the body composition, aerobic power and food intake of sedentary young females in relation to the initial levels of these variables. Thirty one untrained college females (age = 19.8 +/- 0.2 yr, stature = 154.4 +/- 0.8 cm, body mass = 53.3 +/- 1.2 kg, mean +/- SEM) participated in an exercise regimen consisting of 40% of maximum oxygen uptake (VO2max) for 30 minutes per day on a bicycle ergometer 5 times a week in a training period of 12 weeks. Food consumption was ad libitum but the content of daily food intake was recorded accurately throughout the whole training period and analyzed weekly. The average body mass index (BMI) and fat mass relative to body mass (% FM), estimated from the data of skinfold thickness, decreased significantly after the 12 wk training. There were significant negative correlations between the relative changes (% delta s) and initial levels of both body mass (r = -0.447, p < 0.05) and fat mass (r = -0.638, p < 0.05), but the corresponding correlation for lean body mass (LBM) was not significant (r = 0.186, p > 0.05). While the energy intake during the training period did not differ significantly from that during the control period on the average, the % delta value in energy intake between the two periods was negatively correlated to the energy intake during the control period (r = -0.604, p < 0.05). In addition, there were low but significant negative correlations between both the initial levels of BMI and %FM and % delta in energy intake; r = -0.413 (p < 0.05) for BMI and r = -0.393 (p < 0.05) for %FM. However, no significant correlations were found between % delta in energy intake and those in body composition variables (r = 0.116 to 0.237, p > 0.05). On the average VO2max relative to body mass (VO2max/BM) increased significantly, but VO2max relative to LBM (VO2max/LBM) did not. However, not only VO2max/BM but also VO2max/LBM was negatively correlated to the initial level; r = -0.671 (p < 0.05) for VO2max/BM and r = -0.625 for VO2max/LBM. Thus, the present results indicate that whether the body composition, food intake and aerobic power of sedentary young females can be modified by the exercise regimen eliciting 40% of VO2max depends on their initial levels. (+info)
Activity of disulfiram (bis(diethylthiocarbamoyl)disulphide) and ditiocarb (diethyldithiocarbamate) against metronidazole-sensitive and -resistant Trichomonas vaginalis and Tritrichomonas foetus.
Clinical resistance of Trichomonas vaginalis to metronidazole is best correlated with MIC values measured under aerobic conditions. Under these conditions both disulfiram (bis(diethylthiocarbamoyl)disulphide), and its first mammalian metabolite, ditiocarb (diethyldithiocarbamate), showed high levels of activity against metronidazole-sensitive (disulfiram MIC, 0.1-0.7 microM; ditiocarb MIC, 0.3-9 microM) and -resistant (MICs 0.2-1.3 microM and 1.2-9 microM respectively) isolates. Tritrichomonas foetus was also sensitive-the MICs for seven metronidazole-sensitive isolates were 0.1-1.0 microM for disulfiram and 1.0-6.9 microM for ditiocarb; those for two highly metronidazole-resistant strains were 0.3-1.3 microM and 0.6-6 microM respectively. Under anerobic conditions most strains became highly resistant to both compounds. Surprisingly, disulfiram was consistently more active than ditiocarb. (+info)
Acid- and base-induced proteins during aerobic and anaerobic growth of Escherichia coli revealed by two-dimensional gel electrophoresis.
Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified in Escherichia coli. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 proteins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may reverse alkalinization by metabolizing amino acids to produce acidic products. Also induced at high pH, but only in anaerobiosis, was glutamate decarboxylase (GadA). The gad system (GadA/GadBC) neutralizes acidity and enhances survival in extreme acid; its induction during anaerobic growth may help protect alkaline-grown cells from the acidification resulting from anaerobic fermentation. To investigate possible responses to internal acidification, cultures were grown in propionate, a membrane-permeant weak acid which acidifies the cytoplasm. YfiD, a homologue of pyruvate formate lyase, was induced to high levels at pH 4.4 and induced twofold more by propionate at pH 6; both of these conditions cause internal acidification. At neutral or alkaline pH, YfiD was virtually absent. YfiD is therefore a strong candidate for response to internal acidification. Acid or propionate also increased the expression of alkyl hydroperoxide reductase (AhpC) but only during aerobic growth. At neutral or high pH, AhpC showed no significant difference between aerobic and anaerobic growth. The increase of AhpC in acid may help protect the cell from the greater concentrations of oxidizing intermediates at low pH. Isocitrate lyase (AceA) was induced by oxygen across the pH range but showed substantially greater induction in acid or in base than at pH 7. Additional responses observed included the induction of MalE at high pH and induction of several enzymes of sugar metabolism at low pH: the phosphotransferase system components ManX and PtsH and the galactitol fermentation enzyme GatY. Overall, our results indicate complex relationships between pH and oxygen and a novel permeant acid-inducible gene, YfiD. (+info)
Convective oxygen transport and tissue oxygen consumption in Weddell seals during aerobic dives.
Unlike their terrestrial counterparts, marine mammals stop breathing and reduce their convective oxygen transport while performing activities (e.g. foraging, courtship, aggressive interactions, predator avoidance and migration) that require sustained power output during submergence. Since most voluntary dives are believed to remain aerobic, the goal of this study was to examine the potential importance of the dive response in optimizing the use of blood and muscle oxygen stores during dives involving different levels of muscular exertion. To accomplish this, we designed a numerical model based on Fick's principle that integrated cardiac output (Vb), regional blood flow, convective oxygen transport (Q(O2)), muscle oxymyoglobin desaturation and regional rates of oxygen consumption (VO2). The model quantified how the optimal matching or mismatching of QO2 to VO2 affected the aerobic dive limit (ADL). We chose an adult Weddell seal Leptonycotes weddellii on which to base our model because of available data on the diving physiology and metabolism of this species. The results show that the use of blood and muscle oxygen stores must be completed at the same time to maximize the ADL for each level of VO2. This is achieved by adjusting Vb (range 19-94 % of resting levels) and muscle QO2 according to the rate of muscle oxygen consumption (VMO2). At higher values of VMO2, Vb and muscle perfusion must increase to maintain an appropriate QO2/VO2 ratio so that available blood and muscle oxygen stores are depleted at the same time. Although the dive response does not sequester blood oxygen exclusively for brain and heart metabolism during aerobic dives, as it does during forced submersion, a reduction in Vb and muscle perfusion below resting levels is necessary to maximize the ADL over the range of diving VO2 (approximately 2-9 ml O2 min-1 kg-1). Despite the reduction in Vb, convective oxygen transport is adequate to maintain aerobic metabolism and normal function in the splanchnic organs, kidneys and other peripheral tissues. As a result, physiological homeostasis is maintained throughout the dive. The model shows that the cardiovascular adjustments known as the dive response enable the diving seal to balance the conflicting metabolic demands of (1) optimizing the distribution and use of blood and muscle oxygen stores to maximize the ADL over the normal range of diving VO2 and (2) ensuring that active muscle receives adequate oxygen as VMO2 increases. (+info)