The functional significance of glucose dehydrogenase in Klebsiella aerogenes

In order to assess the functional significance of the quinoprotein glucose dehydrogenase recently found to be present in K⁺-limited Klebsiella aerogenes, a broad study was made of the influence of specific environmental conditions on the cellular content of this enzyme. Whereas high activities were manifest in cells from glucose containing chemostat cultures that were either potassium- or phosphate-limited, only low activities were apparent in cells from similar cultures that were either glucose-, sulphate- or ammonia-limited. With these latter two cultures, a marked increase in glucose dehydrogenase activity was observed when 2,4-dinitrophenol (1 mM end concentration) was added to the growth medium. These results suggested that the synthesis of glucose dehydrogenase is not regulated by the level of glucose in the growth medium, but possibly by conditions that imposed an energetic stress upon the cells. This conclusion was further supported by a subsequent finding that K⁺-limited cells that were growing on glycerol also synthesized substantial amounts of glucose dehydrogenase.The enzyme was found to be membrane associated, and preliminary evidence has been obtained that it is located on the periplasmic side of the cytoplasmic membrane and functionally linked to the respiratory chain. This structural and functional orientation is consistent with glucose dehydrogenase serving as a low impedance energy generating system.Abbreviations D dilution rate - DNP 2,4-dinitrophenol - PQQ 2,7,9-tricarboxy-1H-pyrrolo(2,3-f)quinoline-4,5-dione - PTS phosphoenolpyruvate: glucose phosphotransferase - WB Wurster's Blue     

Dual pathways of glycerol assimilation in Klebsiella aerogenes NCIB 418

Klebsiella aerogenes NCIB 418 assimilates glycerol via alternative pathways: one involves a glycerol kinase with a high affinity for glycerol (apparent K _m=1–2×10^–6 M), and the second a glycerol dehydrogenase with a much lower affinity for its substrate (apparent K _m=2–4×10^–2 M).In variously-limited chemostat cultures, one or the other pathway predominated. Thus, aerobic carbonlimited organisms contained only the glycerol kinase pathway whereas aerobic sulphate-limited or ammonia-limited organisms (grown on glycerol) used only the glycerol dehydrogenase pathway. Anaerobic cultures invariably contained glycerol dehydrogenase, and glycerol kinase was absent.Washed suspensions of aerobically-grown organisms oxidized glycerol with kinetics similar to that of the particular enzyme (the primary enzyme of the assimilatory pathway) which they possessed, thus indicating a close association between these two enzymes and the uptake process. But a supply of exogenous glycerol was not a prerequisite for the synthesis of either glycerol kinase or glycerol dehydrogenase, and nor was molecular oxygen the key factor in effecting modulation between the alternative pathways of glycerol metabolism, as had been previously suggested.The physiological significance of dual pathways of glycerol assimilation is discussed.     

The regulation of carbohydrate metabolism in Klebsiella aerogenes NCTC 418 organisms,growing in chemostat culture

Klebsiella aerogenes NCTC 418 was grown in chemostat cultures (D=0.17 hr^-1; pH 6.8; 35° C) that were, successively, carbon-, sulphate-, ammonia-, and phosphate-limited, and which contained as the sole carbon-substrate first glucose, then glycerol, mannitol and lactate. Quantitative analyses of carbon-substrate used and products formed allowed carbon balances to be constructed and direct comparisons to be made of the effciency of substrate utilization. With all sixteen cultures, carbon recoveries of better than 90% were obtained.Optimum utilization of the carbon substrate was invariably found with the carbon-limited cultures, the sole products being organisms and carbon dioxide. But the extent to which excess substrate was over-utilized varied markedly with both the nature of the growth-limitation and the identity of the carbon-substrate. In general, sulphate-, ammonia-, and phosphate-limited cultures utilized glycerol more efficiently than mannitol, mannitol better than lactate, and glucose least efficiently. Glucose-containing cultures also synthesized some extracellular polysaccharide.When the carbon source was in excess, a range of acidic compounds generally were excreted. Sulphate-limited cultures, growing on glucose, excreted much pyruvate and acetate, whereas similarly-limited cultures growing on glycerol, mannitol or lactate produced only acetate. Ammonialimited cultures invariably excreted 2-oxoglutarate and acetate, whereas phosphate-limited cultures produced gluconic acid, 2-ketogluconic acid and acetate, when growing on glucose, but only acetate when growing on mannitol or lactate.From the rates of substrate and oxygen consumption, and the rates of cell synthesis, yield values for both substrate and oxygen were calculated. These showed different trends, but were similar in being highest under carbon-limitation and substantially lower under all other limitations.The physiological significance of these findings, and the probable nature of the regulatory mechanisms underlying overflow metabolism are discussed.     

Production of gluconic acid and 2-ketogluconic acid by Klebsiella aerogenes NCTC 418

2-Ketogluconic acid and, to a lesser extent, gluconic acid were found to be major products of glucose catabolism by phosphate-limited cultures of Klebsiella aerogenes NCTC 418, and together accounted for up to 46% of the glucose carbon that was metabolized.Although the concentrations of both acids increased sub-stantially at low growth rates, their specific rates of synthesis decreased markedly, as did the proportion of glucose converted into these products.Determination of the affinity constant, for glucose, of phosphate-limited organisms showed it to be not significantly different from that of glucose-limited organisms (K _s 50 M), indicative of the phosphotransferase uptake system. And since these organisms possessed an active glucose 6-phosphate dehydrogenase, and had no detectable glucose dehydrogenase activity, it was concluded that gluconic acid and 2-ketogluconic acid arose from their corresponding phosphorylated metabolites, and not directly from glucose.     

Overflow metabolism during anaerobic growth of Klebsiella aerogenes NCTC 418 on glycerol and dihydroxyacetone in chemostat culture

Klebsiella aerogenes NCTC 418 was grown anaerobically in chemostat culture with glycerol as source of carbon and energy. Glycerol-limited cultures did not ferment the carbon source with maximal efficiency but produced considerable amounts of 1,3-propanediol. The fraction of glycerol converted to this product depended on the growth rate and on the limitation: faster growing cells produced relatively more of this compound. Under glycerol excess conditions the energetic efficiency of fermentation was decreased due to the high 1,3-propanediol excretion rate. Evidence is presented that 1,3-propanediol accumulation exerts a profound effect on the cells' metabolic behaviour.When steady state glycerol-limited cultures were instantaneously relieved of the growth limitation a vastly enhanced glycerol uptake rate was observed, accompanied by a shift in the fermentation pattern towards 1,3-propanediol and acetate. This observation was consistent with the extremely high glycerol dehydrogenase activity that was measured in vitro. Some mechanisms that could be responsible for the energy dissipation during this response are discussed.     

Bioenergetic aspects of aerobic growth of Klebsiella aerogenes NCTC 418 in carbon-limited and carbon-sufficient chemostat culture

Carbon-limited chemostat cultures of Klebsiella aerogenes NCTC 418 consumed more oxygen per unit of cell synthesis when growing on mannitol or glycerol than when growing on glucose; and since the maintenance requirements were similar, this suggested that the extra reducing equivalents present in these compounds were oxidized wastefully. By comparison with carbon-limited cultures, carbon-sufficient cultures that were ammonia-, sulphate- or phosphate-limited generally consumed considerably more oxygen per unit of cell synthesis, particularly at low growth rates. Thus, according to the theory of Pirt, these carbon-sufficient cultures had a greatly increased maintenance energy requirement but nevertheless used the remaining energy with a much increased efficiency compared with carbon-limited cultures. This, we suggest, is a false conclusion which stems from the basic assumption that the maintenance requirement does not change with growth rate. Thus we propose an alternative theory which allows for this possibility, and present evidence to show that it may be applicable to both carbon-limited and carbon-sufficient chemostat cultures. Finally we offer an explanation of the high maintenance rate of oxygen consumption found with carbon-sufficient cultures, and consider this phenomenon in relation to the loose coupling between respiration and growth extant in most microbial cultures.     

Isolation and characterization of an alginate lyase from Klebsiella aerogenes

The bacterium Klebsiella aerogenes (type 25) produced an inducible alginate lyase, whose major activity was located intracellularly during all growth phases. The enzyme was purified from the soluble fraction of sonicated cells by ammonium sulfate precipitation, anion- and cation-exchange chromatography and gel filtration. The apparent molecular weight of purified alginate lyase of 28,000 determined by gel filtration and of 31,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the active enzyme was composed of a single polypeptide. The alginate lyase displayed a pH optimum around 7.0 and a temperature optimum around 37°C. The purified enzyme depolymerized alginate by a lyase reaction in an endo manner releasing products which reacted in the thiobarbituric acid assay and absorbed strongly in the ultraviolet region at 235 nm. The alginate lyase was specific for guluronic acidrich alginate preparations. Propylene glycol esters of alginate and O-acetylated bacterial alginates were poorly degraded by the lyase compared with unmodified polysaccharide. The guluronate-specific lyase activity was applied in an enzymatic method to detect mannuronan C-5 epimerase in three different mucoid (alginate-synthesizing) strains of Pseudomonas aeruginosa. This enzyme which converts polymannuronate to alginate could not be demonstrated either extracellularly or intracellularly in all strains suggesting the absence of a polymannuronate-modifying enzyme in P. aeruginosa.Abbreviations poly(ManA) (1–4)--D-mannuronan - poly(GulA) (1–4)--L-guluronan - TBA 2-thiobarbituric acid     

Immunoferritin labeling of respiratory nitrate reductase in membrane vesicles of Bacillus licheniformis and Klebsiella aerogenes

The indirect immunoferritin labeling method was used to localize the membrane-bound respiratory nitrate reductase in membrane vesicles and protoplasts or spheroplasts of Bacillus licheniformis and Klebsiella aerogenes, respectively. For a comparison of the labeling of the various vesicle preparations, which differed not only in size but also in the percentage of inside-out orientation, a quantification of the results was needed to circumvent the problem of non-specifically bound ferritin. From the results the sidedness of the nitrate reductase in the cytoplasmic membrane of the abovementioned bacteria was determined as being cytoplasmic in B. licheniformis and as transmembranous in K. aerogenes.Non-Standard Abbreviations PBS phosphate buffered saline - IgG immunoglobulin G     

Fermentation shifts and metabolic reactivity during anaerobic carbon-limited growth of Klebsiella aerogenes NCTC 418 on fructose,gluconate, mannitol and pyruvate

Klebsiella aerogenes NCTC 418 was grown in chemostat culture under carbon limitation, with fructose, mannitol, gluconate or pyruvate as the growth-limiting substrates, respectively. It was found that under these conditions the carbon sources were fermented with maximal stoichiometry of ATP generation. The Y _ATP values (g cells per mol ATP generated net) were similar for mannitol- and fructose-limited cultures, but gluconate-limited cultures expressed a value that was 20% lower. From these data it was concluded that gluconate-limited cells invest 0.5 ATP in the uptake of 1 gluconate.If the carbon limitation was instantaneously relieved by addition of a saturating amount of the growth-limiting substrate it was found that the response depended on the nature of the carbon substrate, and in particular on the ability to shift the fermentation pattern towards new products. In fructose- and gluconate-limited cultures the specific uptake rate of the carbon source increased substantially, without a concomitant increase in growth rate, and D-lactate appeared as a new fermentation product, in the case of gluconate accompanied by pyruvate. In contrast, with mannitol- and pyruvate-limited cultures the uptake rate of the carbon source and the fermentation pattern did not change. These results are discussed in connection with the functioning of the methylglyoxal by-pass and its role in sustaining metabolic reactivity.     

Influence of growth environment on the phosphoenolpyruvate: Glucose phosphotransferase activities of Escherichia coli and Klebsiella aerogenes: A comparative study

A consistent difference was found between glucose-limited cultures of Escherichia coli and Klebsiella aerogenes strains in the manner which their apparent cellular content of glucose: phosphoenolpyruvate phosphotransferase (glucose-PTS) varied with growth rate. With the former strains, activity increased as a function of growth rate; in the latter it decreased. However, under glucose-sufficient conditions (potassium-or ammonia-limitation) both species behaved similarly; the glucose-PTS activity was lower and bore no obvious relationship to the rate of glucose consumption expressed by the growing culture. These results are discussed in relation to the role of glucose as a regulator of glucose-PTS synthesis, and to the likely contribution which the glucose-PTS makes to the overall rate of glucose uptake, particularly by cells growing in glucose-sufficient environments.Abbreviation Glucose-PTS phosphoenolpyruvate phosphotransferase From May to November 1978 on study leave in the University of Amsterdam     

Contributions of apoplasmic cadmium accumulation,antioxidative enzymes and induction of phytochelatins in cadmium tolerance of the cadmium-accumulating cultivar of black oat (<Emphasis Type="Italic">Avena strigosa</Emphasis> Schreb.)

The contributions of cadmium (Cd) accumulation in cell walls, antioxidative enzymes and induction of phytochelatins (PCs) to Cd tolerance were investigated in two distinctive genotypes of black oat (Avena strigosa Schreb.). One cultivar of black oat ‘New oat’ accumulated Cd in the leaves at the highest concentration compared to another black oat cultivar ‘Soil saver’ and other major graminaceous crops. The shoot:root Cd ratio also demonstrated that ‘New oat’ was the high Cd-accumulating cultivar, whereas ‘Soil saver’ was the low Cd-accumulating cultivar. Varied levels of Cd exposure demonstrated the strong Cd tolerance of ‘New oat’. By contrast, low Cd-accumulating cultivar ‘Soil saver’ suffered Cd toxicity such as growth defects and increased lipid peroxidation, even though it accumulated less Cd in shoots than ‘New oat’. Higher activities of ascorbate peroxidase (EC 1.11.1.11) and superoxide dismutase (EC 1. 15. 1. 1) were observed in the leaves of ‘New oat’ than in ‘Soil saver’. No advantage of ‘New oat’ in PCs induction was observed in comparison to Cd-sensitive cultivar ‘Soil saver’, although Cd exposure increased the concentration of total PCs in both cultivars. Higher and increased Cd accumulation in cell wall fraction was observed in shoots of ‘New oat’. On the other hand, in ‘Soil saver’, apoplasmic Cd accumulation showed saturation under higher Cd exposure. Overall, the present results suggest that cell wall Cd accumulation and antioxidative activities function in the tolerance against Cd stress possibly in combination with vacuolar Cd compartmentation.     

A comparison of alternate metabolic strategies for the utilization of D-arabinose

Summary Mutants ofKlebsiella aerogenes W70 that metabolize the uncommon pentose D-arabinose were isolated. These mutants were found to be either constitutive or indicible by D-arabinose for the synthesis of enzymes in the L-fucose pathway. Such mutants could then utilize L-fucose isomerase to convert the structurally similar D-arabinose molecule to D-ribulose. D-Ribulose is an inter-mediate and the inducer of an existing ribitol pathway and could thus be metabolized. In those D-arabinose-positive mutants where the ribitol pathway was blocked by mutation, D-ribulose could alternatively be metabolized by using the remaining L-fucose pathway enzymes. When the two D-arabinose catabolic routes were compared, catabolism of D-arabinose via the ribitol pathway was found to be more efficient. Catabolism of D-arabinose using the L-fucose pathway per-mitted D-ribulose to escape into the media and produced an unmetabolizable end product, L-glycolic acid. A comparison of growth using constitutive versus inducible control of the borrowed L-fucose isomerase did not reveal an advantage for one control type over the other. Several differences were observed,however, when we determined the degree to which these control mutations perturbed the normal functioning of the L-fucose and associated pathways. Growth of the constitutive mutant was impaired with L-fucose as substrate. The inducible-control mutant had altered growth characteristics on ribitol and L-rhamnose.     

Effect of nano cadmium sulfide on the electron transfer reactivity and peroxidase activity of hemoglobin

Hemoglobin (Hb) is immobilized with cadmium sulfide (CdS) nanoparticles (NPs) on pyrolytic graphite (PG) electrode to characterize the electrochemical reactivity and peroxidase activity of the protein. The result demonstrates that fine redox waves of Hb can be achieved after this protein is entrapped in CdS NPs. Meanwhile, the protein can exhibit nice catalytic activity towards hydrogen peroxide (H2O2). Linear relationship between the reductive peak current and the H2O2 concentration has been obtained from 5.0 x 10(-6) to 4.0 x 10(-4) mol/L, on the basis of which a new kind of H2O2 biosensor might be developed in the future.     

Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene

Microbial production of butanediol and acetoin has received increasing interest because of their diverse potential practical uses. Although both products are fermentative in nature, their optimal production requires a low level of oxygen. In this study, the use of a recombinant oxygen uptake system on production of these metabolites was investigated. Enterobacter aerogenes was transformed with a pUC8-based plasmid carrying the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). The presence of vgb and production of VHb by this strain resulted in an increase in viability from 72 to 96 h in culture, but no overall increase in cell mass. Accumulation of the fermentation products acetoin and butanediol were enhanced (up to 83%) by the presence of vgb/VHb. This vgb/VHb related effect appears to be due to an increase of flux through the acetoin/butanediol pathway, but not at the expense of acid production.     

Deletion analysis of the nitrogen fixation regulatory gene,nifL of Klebsiella pneumoniae

A number of in-frame deletions have been constructed in the Klebsiella pneumoniae regulatory gene nifL. The effects of each nifL mutation on NifA-mediated expression from the nifH promoter of K. pneumoniae have then been assessed with respect to both nitrogen and oxygen control. These experiments indicate that, in contrast to the situation with the homologous regulatory proteins NtrB and NtrC, NifA activity is not impaired in the absence of NifL. We conclude that the only function of NifL is to inactivate NifA in response to an increase in the nitrogen or oxygen status of the cell.     

Energetic aspects of anaerobic growth of Aerobacter aerogenes in complex medium

Molar growth yields for anaerobic growth of Aerobacter aerogenes in complex medium were much higher than for growth in minimal medium. In batch cultures the molar growth yield for glucose varied from 44 to 50 and Y _ATP from 17.1 to 18.8. For glucose-limited chemostat cultures a value of 17.5 g/mole was found for Y _ATP ^max and a value of 2.3 mmoles ATP/g dry weight h for the maintenance coeficient. Growth dependent pH changes were used to control the addition of fresh medium, containing excess of glucose to a continuous culture. The specific growth rate and the population density were dependent on the pH difference between the inflowing medium and the culture. At a value of 1.44 h^-1 the molar growth yield for glucose was about 70 and Y _ATP about 28.5. An-equation is presented, which gives the relation between theoretical and experimental Y _ATP ^max values.     

A new method to detect cadmium uptake in protoplasts

The mechanism for cadmium (Cd²⁺) uptake into the cytosol of protoplasts from 5- to 7-day-old wheat seedlings (Triticum aestivum L. cv. Kadett) was investigated by a new method, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. Cadmium fluorescence gradually increased in the cytosol of shoot and root protoplasts upon repeated additions of CdCl₂ to the external medium, reflecting an uptake of Cd²⁺. The uptake was inhibited by calcium and potassium chloride, as well as by Verapamil and tetraethylammonium (TEA), inhibitors of calcium and potassium channels, respectively. Calcium competitively inhibited the cadmium uptake. The metabolic inhibitors vanadate and dinitrophenol partly inhibited the uptake, suggesting it was dependent on membrane potential. The results indicate that cadmium is taken up by channels permeable to both calcium and potassium. The total uptake of cadmium into the protoplasts was also detected by unidirectional flux analyses using ¹⁰⁹Cd²⁺, and showed approximately the same maximal concentration of Cd²⁺ as the fluorescence measurements. By combining the two methods it is possible to detect both uptake into the cytosol and into the vacuole.Abbreviations BTC-5N, AM Acetoxymethyl ester of 5-nitrobenzothiazole coumarin - DNP 2,4-Dinitrophenol - TEA Tetraethylammonium     

Studying the ability to absorb heavy metal of cadmium on the amount of sugar and chlorophyll using seedlings of berry specie (Morus alba) in pollution area

Heavy metals are from the group of problematic factors in the ecosystem that due to being non-absorbable and having physiological effects on the activity of living organisms at low concentrations are of particular importance. In this study, the ability of berry specie to absorb heavy metal of cadmium was studied. One year old seedlings of berry specie were prepared from the nursery, concentrations of zero and 100 mg per liter of cadmium chloride solution were added to the seedlings pots soil after calculation, and after a three-month period of seedling growth, shoots and roots were separated, then the concentration amount of cadmium in the samples was determined and data were examined. The results of data analysis showed that the highest rate of cadmium accumulation in stems, roots and soil in treatment concentration is 54.76, 107.75 and 8.825 mg per kg, respectively, and the rate of cadmium accumulation in total chlorophyll and sugar in treatment concentration is 3.16 and 0.6693 mg per g, respectively, and based on the results of this research berry specie is relatively suitable for remediation of soils contaminated with cadmium.