Suppression of Photoreactivating Enzyme Production in Escherichia coli Grown Under Anaerobic Conditions

Studies with two K-12 mutants of Escherichia coli have indicated that the production of photoreactivating enzyme is suppressed under anaerobic growth conditions. Under no condition tested could the estimated number of photoreactivating enzyme molecules per cell be reduced below an average of one.     

Siderophore Production by Pseudomonas stutzeri under Aerobic and Anaerobic Conditions

The siderophore production of the facultative anaerobe Pseudomonas stutzeri, strain CCUG 36651, grown under both aerobic and anaerobic conditions, was investigated by liquid chromatography and mass spectrometry. The bacterial strain has been isolated at a 626-m depth at the Äspö Hard Rock Laboratory, where experiments concerning the geological disposal of nuclear waste are performed. In bacterial culture extracts, the iron in the siderophore complexes was replaced by gallium to facilitate siderophore identification by mass spectrometry. P. stutzeri was shown to produce ferrioxamine E (nocardamine) as the main siderophore together with ferrioxamine G and two cyclic ferrioxamines having molecular masses 14 and 28 atomic mass units lower than that of ferrioxamine E, suggested to be ferrioxamine D₂ and ferrioxamine X₁, respectively. In contrast, no siderophores were observed from anaerobically grown P. stutzeri. None of the siderophores produced by aerobically grown P. stutzeri were found in anaerobic natural water samples from the Äspö Hard Rock Laboratory.     

Reduction of Soluble and Insoluble Iron Forms by Membrane Fractions of Shewanella oneidensis Grown under Aerobic and Anaerobic Conditions

The effect of iron substrates and growth conditions on in vitro dissimilatory iron reduction by membrane fractions of Shewanella oneidensis MR-1 was characterized. Membrane fractions were separated by sucrose density gradients from cultures grown with O₂, fumarate, and aqueous ferric citrate as the terminal electron acceptor. Marker enzyme assays and two-dimensional gel electrophoresis demonstrated the high degree of separation between the outer and cytosolic membrane. Protein expression pattern was similar between chelated iron- and fumarate-grown cultures, but dissimilar for oxygen-grown cultures. Formate-dependent ferric reductase activity was assayed with citrate-Fe³⁺, ferrozine-Fe³⁺, and insoluble goethite as electron acceptors. No activity was detected in aerobic cultures. For fumarate and chelated iron-grown cells, the specific activity for the reduction of soluble iron was highest in the cytosolic membrane. The reduction of ferrozine-Fe³⁺ was greater than the reduction of citrate-Fe³⁺. With goethite, the specific activity was highest in the total membrane fraction (containing both cytosolic and outer membrane), indicating participation of the outer membrane components in electron flow. Heme protein content and specific activity for iron reduction was highest with chelated iron-grown cultures with no heme proteins in aerobically grown membrane fractions. Western blots showed that CymA, a heme protein involved in iron reduction, expression was also higher in iron-grown cultures compared to fumarate- or aerobic-grown cultures. To study these processes, it is important to use cultures grown with chelated Fe³⁺ as the electron acceptor and to assay ferric reductase activity using goethite as the substrate.     

Effect of Oxygen on Several Enzymes Involved in the Aerobic and Anaerobic Utilization of Glucose in Escherichia coli

By using the continuous culture technique, the transition from aerobiosis to anaerobiosis and its effect on a number of enzymes has been investigated in Escherichia coli K-12. A decrease in the oxygen partial pressure below 28.0 mm of Hg resulted firstly in an increase of the respiratory enzymes (reduced nicotinamide adenine dinucleotide [NADH] oxidase, 2.53-fold; succinic dehydrogenase, 1.4-fold; cytochrome b(1), 3.91-fold; and cytochrome a(2), 2.45-fold) before the electron transport system gradually collapsed as cytochrome a(2), followed by cytochrome b(1), succinic dehydrogenase, and finally NADH oxidase decreased in activity. The change from respiration to fermentation was initiated well before the oxygen tension reached zero by the increase in levels of fructose diphosphate-aldolase, glucose 6-phosphate, and 6-phosphogluconate dehydrogenases and a decrease in 2-oxoglutarate dehydrogenase. Whem the dissolved oxygen tension reached zero, dry weight and CO(2) formation together with isocitrate dehydrogenase decreased, whereas acid production and phosphofructokinase synthesis started to increase. Enzymatic investigations revealed that the kinetics of the enzyme phosphofructokinase from strict aerobic cultures (6.9 ppm oxygen in solution) was adenosine triphosphate (ATP)-insensitive, whereas the same enzyme from anaerobic cultures was ATP-sensitive. A mechanism is proposed for the change from aerobiosis to anaerobiosis together with the occurring change in glucose regulation.     

Amylolytic Activities in Cereal Seeds under Aerobic and Anaerobic Conditions

An adequate carbohydrate supply contributes to the survival of seeds under conditions of limited oxygen availability. The amount of soluble, readily fermentable carbohydrates in dry cereal seeds is usually very limited, with starch representing the main storage compound. Starch breakdown during the germination of cereal seeds is the result of the action of hydrolytic enzymes and only through the concerted action of [alpha]-amylase (EC 3.2.1.1), [beta]-amylase (EC 3.2.1.2), debranching enzyme (EC 3.2.1.41), and [alpha]-glucosidase (EC 3.2.1.20) can starch be hydrolyzed completely. We present here data concerning the complete set of starch-degrading enzymes in three cereals, rice (Oryza sativa L.), which is tolerant to anaerobiosis, and wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), which are unable to germinate under anoxia. Among the cereal seeds tested under anoxia, only rice is able to degrade nonboiled, soluble starch, reflecting the ability to degrade the starch granules in vivo. This is explained by the presence of the complete set of enzymes needed to degrade starch completely either as the result of de novo synthesis ([alpha]-amylase, [beta]-amylase) or activation of preexisting, inactive forms of the enzyme (debranching enzyme, [alpha]-glucosidase). These enzymes are either absent or inactive in wheat and barley seeds kept under anaerobic conditions.     

The Influence of Nitroxyl on Escherichia coli Cells Grown under Carbonyl Stress Conditions

Applied Biochemistry and Microbiology - Carbonyl stress is an increase in the amount of monosaccharides and active dicarbonyl compounds (glyoxal and methylglyoxal (MG)), which leads to raising the...     

Attachment Behavior of Shewanella putrefaciens onto Magnetite under Aerobic and Anaerobic Conditions

In this study, batch experiments were used to characterize attachment behavior of Shewanella putrefaciens strain 200R to ferrihydrite and magnetite. Attachment was quantified in batch experiments with a 0.01 M NaNO ₃ solution as a function of pH (ranging from 3 to 10), sorbed anion (PO₄ ^{3 ?} ), and growth conditions (aerobic vs. anaerobic). Electrophoretic mobility data was collected for S. putrefaciens cells and magnetite grains and used as a means to interpret the role of electrostatic interaction in attachment studies. Little difference in attachment behavior was observed as a function of growth conditions or surface treatments. The exception was at pH ranging from 2 to 4, under anaerobic conditions, where increased attachment was measured on magnetite surfaces with sorbed PO₄ ^{3 ?} . This increased attachment was attributed to development of Fe-PO₄ surface complexes or secondary mineral phases, resulting in altered surface interactions between cell and mineral surfaces. Attachment was irreversible and increased with time under anaerobic conditions even under elevated pH conditions unfavourable to electrostatic interactions between cells and mineral surfaces. These results suggest that electrophoretic mobility data in this system is not a good predictor of attachment behavior, while surface charge development via protonation and deprotonation of surface functional groups is consistent with experimental attachment data. In this study, S. putrefaciens appears to utilize polymers or pili to remain attached to Fe-oxides and this process may facilitate Fe reduction on these surfaces. Results from this study underscore the need for quantitative bulk measurements of microbial attachment to accurately predict partitioning of dissimilatory iron reducing bacteria between solution and solid phases.     

Oxygen-Insensitive Nitroreductases NfsA and NfsB of Escherichia coli Function under Anaerobic Conditions as Lawsone-Dependent Azo Reductases

Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E. coli and thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.     

Interactions of Tetrahymena pyriformis, Escherichia coli, Azotobacter vinelandii, and Glucose in a Minimal Medium

A study was made of the food web formed from a protozoon, two bacteria, and a glucose minimal medium in chemostat culture. The system was also divided into simpler parts, first by omitting the protozoon to obtain a competition system, and then by omitting one or the other of the bacteria to obtain two food chains. In the competition studies, one bacterium was displaced by the other at all holding times used. In the food chain studies, sustained oscillations of the population densities of predator and prey developed at short holding times, and then changed to damped oscillations at longer holding times. In addition, the level of residual glucose remained high at long holding times. A new model of microbial growth is necessary to explain these results. In the food web studies, predation of the protozoon on the two bacteria stabilized the competition between the latter and allowed their coexistence in the same habitat. Thus, Gause's principle was circumvented.     

Mutants of Escherichia coli Defective in Ribonucleoside and Deoxyribonucleoside Catabolism

From Escherichia coli B, mutants were prepared that lacked the enzymes adenosine deaminase, cytidine deaminase, and purine nucleoside phosphorylase. In each case, the mutant lacked enzyme activity for both ribonucleoside and deoxyribonucleoside. Mutants lacking purine nucleoside phosphorylase lost the capacity to cleave the nucleosides of adenine, guanine, and hypoxanthine.     

Catabolism of phenylpropionic acid and its 3-hydroxy derivative by Escherichia coli

A number of laboratory strains and clinical isolates of Escherichia coli utilized several aromatic acids as sole sources of carbon for growth. E. coli K-12 used separate reactions to convert 3-phenylpropionic and 3-(3-hydroxyphenyl)propionic acids into 3-(2,3-dihydroxyphenyl)propionic acid which, after meta-fission of the benzene nucleus, gave succinate, pyruvate, and acetaldehyde as products. Enzyme assays and respirometry showed that all enzymes of this branched pathway were inducible and that syntheses of enzymes required to convert the two initial growth substrates into 3-(2,3-dihydroxyphenyl)propionate are under separate control. E. coli K-12 also grew with 3-hydroxycinnamic acid as sole source of carbon; the ability of cells to oxidize cinnamic and 3-phenylpropionic acids, and hydroxylated derivatives, was investigated. The lactone of 4-hydroxy-2-ketovaleric acid was isolated from enzymatic reaction mixtures and its properties, including optical activity, were recorded.     

Biocalorimetric Studies of the Metabolic Activity of Pseudomonas aeruginosa Aerobically Grown in a Glucose-Limited Complex Growth Medium

Biocalorimetric experiments were performed to investigate the aerobic growth of Pseudomonas aeruginosa, isolated from tannery saline wastewater. Growth factors (pH, Inoculum size, carbon source, temperature, aeration rate, and agitation rate) were optimized in shaker and calorimeter based on the growth of P. aeruginosa and heat generation rates. A limiting value of 0.2% glucose concentration was found to be optimum for the growth of P. aeruginosa in a complex growth medium, and the heat flux (q_r) profiles resulting from the metabolic activity of P. aeruginosa further confirmed this observation. The bacterial growth profile was found to correlate well with the metabolic heat generated. Heat-yield values were calculated for both glucose consumption and the growth of P. aeruginosa from the calorimetric results. Metabolic shifts in substrate uptake from glucose to peptone present in growth medium was observed by the variations in heat-flux profile. The calorimetric data presented in this study should be useful in understanding the behavior of the isolated bacterial strain in degrading complex and mixed substrates commonly observed in tannery saline waste stream, and further to extend the results for scale-up studies.     

Arginine Catabolism and the Arginine Succinyltransferase Pathway in Escherichia coli

Arginine catabolism produces ammonia without transferring nitrogen to another compound, yet the only known pathway of arginine catabolism in Escherichia coli (through arginine decarboxylase) does not produce ammonia. Our aims were to find the ammonia-producing pathway of arginine catabolism in E. coli and to examine its function. We showed that the only previously described pathway of arginine catabolism, which does not produce ammonia, accounted for only 3% of the arginine consumed. A search for another arginine catabolic pathway led to discovery of the ammonia-producing arginine succinyltransferase (AST) pathway in E. coli. Nitrogen limitation induced this pathway in both E. coli and Klebsiella aerogenes, but the mechanisms of activation clearly differed in these two organisms. We identified the E. coli gene for succinylornithine aminotransferase, the third enzyme of the AST pathway, which appears to be the first of an astCADBE operon. Its disruption prevented arginine catabolism, impaired ornithine utilization, and affected the synthesis of all the enzymes of the AST pathway. Disruption of astB eliminated succinylarginine dihydrolase activity and prevented arginine utilization but did not impair ornithine catabolism. Overproduction of AST enzymes resulted in faster growth with arginine and aspartate. We conclude that the AST pathway is necessary for aerobic arginine catabolism in E. coli and that at least one enzyme of this pathway contributes to ornithine catabolism.