The effect of<Emphasis Type="Italic"> pfl</Emphasis> gene knockout on the metabolism for optically pure <Emphasis Type="SmallCaps">d</Emphasis>-lactate production by<Emphasis Type="Italic"> Escherichia coli</Emphasis>

The effect of gene knockout on metabolism in the pflA^–, pflB^–, pflC^–, and pflD^– mutants of Escherichia coli was investigated. Batch cultivations of the pfl ^– mutants and their parent strain were conducted using glucose as a carbon source. It was found that pflA^– and pflB^– mutants, but not pflC^– and pflD^– mutants, produced large amounts of d-lactate from glucose under the microaerobic condition, and the maximum yield was 73%. In order to investigate the metabolic regulation mechanism, we measured enzyme activities for the following eight enzymes: glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase, lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, acetate kinase, and alcohol dehydrogenase. Intracellular metabolite concentrations of glucose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate, pyruvate, acetyl coenzyme A as well as ATP, ADP, AMP, NADH, and NAD⁺ were also measured. It was shown that the GAPDH and LDH activities were considerably higher in pflA^– and pflB^– mutants, which implies coupling between NADH production and consumption between the two corresponding reactions. The urgent energy requirement was shown by the lower ATP/AMP level due to both oxygen limitation and pfl gene knockout, which promoted significant stepping-up of glycolysis when using glucose as a carbon source. It was shown that the demand for energy is more important than intracellular redox balance, thus excess NADH produced through GAPDH resulted in a significantly higher intracellular NADH/NAD⁺ ratio in pfl ^– mutants. Consequently, the homolactate production was achieved to meet the requirements of the redox balance and the energy production through glycolysis. The effect of using different carbon sources such as gluconate, pyruvate, fructose, and glycerol was investigated.     

Metabolic regulation analysis of <Emphasis Type="Italic">icd</Emphasis>-gene knockout <Emphasis Type="Italic">Escherichia coli</Emphasis> based on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements

An integrated study of cell growth characteristics, enzyme activities and protein expression patterns was carried out to investigate how the central metabolism of Escherichia coli changes upon knockout of the isocitrate dehydrogenase (ICDH) gene (icd) in the tricarboxylic acid cycle. Deletion of the icd gene led to reduced specific growth rate and reduced specific glucose consumption rate. The reduced specific growth rate in the icd mutant was due mainly to the lower intracellular ATP/ADP ratio as well as to the lower NADPH/NADP⁺ ratio compared with those in the parent strain. However, the specific carbon dioxide evolution rate was found to be higher in the icd mutant strain compared to the parent E. coli. This may be due to the higher activity of 6-phosphogluconate dehydrogenase, phosphoenol pyruvate carboxykinase and NADP⁺-dependent malic enzymes. The glyoxylate pathway was also utilized, as evidenced by the significant upregulation of isocitrate lyase and malate synthase activity in the icd mutant E. coli. The appearance of the glyoxylate pathway caused lower acetate production. Of 21 proteins showing altered expression levels, 17 were successfully identified with the aid of MALDI-TOF mass spectrometry. The results showed that the abolition of ICDH activity significantly affected the respiratory system and electron transport chain, as evidenced by the significant downregulation of proteins encoded by the genes nuoE, nuoH, cydA and cyoA in icd mutant E. coli compared to the parent.     

Production of isoamyl acetate in<Emphasis Type="Italic"> ackA-pta</Emphasis> and/or<Emphasis Type="Italic"> ldh</Emphasis> mutants of<Emphasis Type="Italic"> Escherichia coli</Emphasis> with overexpression of yeast<Emphasis Type="Italic"> ATF2</Emphasis>

The gene coding for alcohol acetyltransferase (ATF2), which catalyzes the esterification of isoamyl alcohol and acetyl coenzyme A (acetyl-CoA), was cloned from Saccharomyces cerevisiae and expressed in Escherichia coli. This genetically engineered strain of E. coli produced the ester isoamyl acetate when isoamyl alcohol was added externally to the cell culture medium. Various competing pathways at the acetyl-CoA node were inactivated to increase the intracellular acetyl-CoA pool and divert more carbon flux to the ester synthesis pathway. Several strains with deletions in the ackA-pta and/or ldh pathways and bearing the ATF2 on a high-copy-number plasmid were constructed and studied. Compared to the wild-type, ackA-pta and nuo mutants produced higher amounts of ester and an ackA-pta-ldh-nuo mutant lower amounts. Isoamyl acetate production correlated well with intracellular coenzyme A (CoA) and acetyl-CoA levels. The ackA-pta-nuo mutant had the highest intracellular CoA/acetyl-CoA level and hence produced the highest amount of ester (1.75 mM) during the growth phase under oxic conditions and during the production phase under anoxic conditions.     

Effect of <Emphasis Type="Italic">poxB</Emphasis> gene knockout on metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis> based on growth characteristics and enzyme activities

The effect of poxB gene knockout on metabolism in Escherichia coli was investigated in the present paper based on the growth characteristics and the activities of the enzymes involved in the central metabolic pathways. The absence of pyruvate oxidase reduced the glucose uptake rate and cell growth rate, and increased O₂ consumption and CO₂ evolution. The enzyme assay results showed that although glucokinase activity increased, the flux through glycolysis was reduced due to the down-regulation of the other glycolytic enzymes such as 6-phosphofructosekinase and fructose bisphosphate aldolase in the poxB mutant. TCA cycle enzymes such as citrate synthase and malate dehydrogenase were repressed in the poxB mutant when the cells were cultivated in LB medium. The pyruvate oxidase mutation also resulted in the activation of glucose-6-phosphate dehydrogenase and acetyl-CoA synthetase. All these results suggest that pyruvate oxidase is not only a stationary-phase enzyme as previously known, and that the removal of the poxB gene affects the central metabolism at the enzyme level in E. coli.     

Control of pyrimidine nucleotide formation in <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fulva</Emphasis>

Control of pyrimidine formation was examined in Pseudomonas fulva ATCC 31418. Pyrimidine supplementation lowered pyrimidine biosynthetic pathway enzyme activities in cells grown on glucose or succinate as a carbon source indicating possible repression of enzyme synthesis. Pyrimidine limitation experiments were conducted using an orotidine 5′-monophosphate decarboxylase mutant strain isolated in this study. Compared to uracil-supplemented, glucose-grown mutant cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities to increase about 6-, 13-, 3-, 15-fold, respectively, which confirmed regulation of enzyme synthesis by pyrimidines. At the level of enzyme activity, transcarbamoylase activity in Ps. fulva was strongly inhibited by pyrophosphate, CTP, GTP and GDP under saturating substrate concentrations.     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

Effect of <Emphasis Type="Italic">cra</Emphasis> gene knockout together with <Emphasis Type="Italic">edd</Emphasis> and <Emphasis Type="Italic">iclR</Emphasis> genes knockout on the metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved. Dayanidhi Sarkar and Khandaker Al Zaid Siddiquee have contributed equally.     

Effect of salt stress on physiological and antioxidative responses in two species of <Emphasis Type="Italic">Salicornia</Emphasis> (<Emphasis Type="Italic">S. persica</Emphasis> and <Emphasis Type="Italic">S. europaea</Emphasis>)

The effects of salt stress on growth parameters, free proline content, ion accumulation, lipid peroxidation, and several antioxidative enzymes activities were investigated in S. persica and S. europaea. The seedlings were grown for 2 months in half-strength Hoagland solution and treated with different concentrations of NaCl (0, 85, 170, 340, and 510 mM) for 21 days. The fresh and dry weights of both species increased significantly at 85 and 170 mM NaCl and decreased at higher concentrations. Salinity increased proline content in both the species as compared to that of control. Sodium (Na⁺) content in roots and shoots increased, whereas K⁺ and P_i content in both organs decreased. At all NaCl concentrations, the total amounts of Na⁺ and K⁺ were higher in shoots than in roots. Malondialdehyde (MDA) content declined at moderate NaCl concentrations (85 and 170 mM) and increased at higher levels. With increased salinity, superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPX) activities also increased gradually in both species. In addition, it seems that GPX, CAT, and SOD activities play an essential protective role in the scavenging reactive oxygen species (ROS) in both species. Native polyacrylamide gel electrophoresis (PAGE) indicated different isoform profiles between S. persica and S. europaea concerning antioxidant enzymes. These results showed that S. persica exhibits a better protection mechanism against oxidative damage and it is more salt-tolerant than S. europaea possibly by maintaining and/or increasing growth parameters, ion accumulation, and antioxidant enzyme activities.     

Allelopathic impact of artificially applied coumarin on <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">niveum</Emphasis>

Watermelon production is threatened by fusarium wilt caused by Fusarium oxysporum f.sp. niveum (FON) in continuous cultivation system. Some elements, mainly allelochemicals, released from living roots or decayed plants might be associated with the disease. The purpose of this work was to evaluate the possible impact of coumarin, one kind of watermelon allelochemical, on FON. Furthermore, possible new mechanisms might be investigated during the ecological interactions of plant-microbe. Results showed that coumarin strongly inhibited growth of FON leading to a decrease in its biomass, dry weight of mycelia of FON in a liquid culture. The dry weight was decreased by 62.9% compared with control. The hyphal growth of FON on plates was stopped at high (>400 mg l⁻¹) concentrations of coumarin. At 320 mg l⁻¹, sporulation and enzyme activities of FON were also severely suppressed by coumarin. The yield of conidia, and the activities of proteinase, cellulase, and amylase were reduced by 98.9%, 79.7%, 29.8% and 15.9% respectively. However, conidial germination and mycotoxin (MT) production of FON were greatly stimulated, being increased by 55.7% and 14.9 fold at 320 mg l⁻¹ respectively. We conclude that coumarin acted as an allelochemical substance to inhibit growth and pathogenic enzyme activities of FON but to stimulate mycotoxin production and conidial germination. It was suggested that coumarin acted as a signal transduction element bridging plant and pathogen in the process of plant-microbe interactions.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Chemoorganoheterotrophic Growth of <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis> and <Emphasis Type="Italic">Nitrosomonas eutropha</Emphasis>

The ammonia oxidizers Nitrosomonas europaea and Nitrosomonas eutropha are able to grow chemoorganotrophically under anoxic conditions with pyruvate, lactate, acetate, serine, succinate, α-ketoglutarate, or fructose as substrate and nitrite as terminal electron acceptor. The growth yield of both bacteria is about 3.5 mg protein (mmol pyruvate)⁻¹ and the maximum growth rates of N. europaea and N. eutropha are 0.094 d⁻¹ and 0.175 d⁻¹, respectively. In the presence of pyruvate and CO₂ about 80% of the incorporated carbon derives from pyruvate and about 20% from CO₂. Pyruvate is used as energy and only carbon source in the absence of CO₂ (chemoorganoheterotrophic growth). CO₂ stimulates the chemoorganotrophic growth of both ammonia oxidizers and the expression of ribulose bisphosphate carboxylase/oxygenase is down-regulated at increasing CO₂ concentration. Ammonium, although required as nitrogen source, is inhibitory for the chemoorganotrophic metabolism of N. europaea and N. eutropha. In the presence of ammonium pyruvate consumption and the expression of the genes aceE, ppc, gltA, odhA, and ppsA (energy conservation) as well as nirK, norB, and nsc (denitrification) are reduced.     

Ethylene biosynthesis in a chilling-sensitive<Emphasis Type="Italic">Arabidopsis</Emphasis> mutant,<Emphasis Type="Italic">chs4-2</Emphasis>

We investigated chilling-induced changes in ethylene levels in Arabidopsis to find plants with distinct patterns of ethylene production in the cold-related biosynthetic pathway. The sensitive mutants identified here includedchs1-2,chs4-2, andchs6-2. Among these, plants of thechs4-2 mutant produced more ethylene than did the wild type after both were transferred from 4°C or 10°C to 22°C. This mutant also showed less freezing tolerance and more electrolyte leakage than the wild-type plants. Our results suggest a relationship between ethylene biosynthesis and chilling sensitivity in the mutant To determine which of the enzymes involved in ethylene biosynthesis were induced by chilling, we tested the activities of ACC synthase and ACC oxidase in both mutant and wild-type plants, and found greater activity by ACC synthase as well as a higher ACC content in the mutants after all the plants were transferred from 10°C to 22°C. However, ACC oxidase activity did not differ between mutant and wild-type plants in response to chilling treatment Therefore, we conclude thatchs4-2 mutants produce more ethylene than do other mutants or the wild type during their recovery from chilling conditions. Furthermore, we believe that ACC synthase is the key enzyme involved in this response.     

Effect of modified glucose catabolism on xanthan production in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

In this study, the glucose 6-phosphate dehydrogenase gene (XOO2314) was inactivated in order to modulate the intracellular glucose 6-phosphate, and its effects on xanthan production in a wild-type strain of Xanthomonas oryzae were evaluated. The intracellular glucose 6-phosphate was increased from 17.6 to 99.4 μmol g⁻¹ (dry cell weight) in the gene-disrupted mutant strain. The concomitant increase in the glucose 6-phosphate was accompanied by an increase in xanthan production of up to 2.23 g l⁻¹ (culture medium). However, in defined medium supplemented with 0.4% glucose, the growth rate of the mutant strain was reduced to 52.9% of the wild-type level. Subsequently, when a family B ATP-dependent phosphofructokinase from Escherichia coli was overexpressed in the mutant strain, the growth rate was increased to 142.9%, whereas the yields of xanthan per mole of glucose remained approximately the same.     

Identification of a novel UDP-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine enolpyruvyl transferase (MurA) from <Emphasis Type="Italic">Vibrio fischeri</Emphasis> that confers high fosfomycin resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis>

MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 μg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.     

The importance of<Emphasis Type="Italic"> porE</Emphasis> and<Emphasis Type="Italic"> porF</Emphasis> in the anabolic pyruvate oxidoreductase of<Emphasis Type="Italic"> Methanococcus maripaludi</Emphasis>s

The operon of the anabolic pyruvate oxidoreductase (POR) of Methanococcus maripaludis encodes two genes (porEF) whose functions are unknown. Because these genes possess sequence similarity to polyferredoxins, they may be electron carriers to the POR. To elucidate whether the methanococcal POR requires PorEF for activity, a deletion mutant, strain JJ150, lacking porEF was constructed. Compared to the wild-type strain JJ1, the mutant grew more slowly in minimal medium and minimal plus acetate medium, and pyruvate-dependent methanogenesis was inhibited. In contrast, the methyl-viologen-dependent pyruvate-oxidation activity of POR, carbon monoxide dehydrogenase, and hydrogenase activities of the mutant were similar to those of the wild-type. Upon genetic complementation of the mutant with porEF in the methanococcal shuttle vector pMEV2+porEF, growth in minimal medium and pyruvate-dependent methanogenesis were restored to wild-type levels. Complementation with porE alone restored methanogenesis from pyruvate but not growth in minimal medium. Complementation with porF alone partially restored growth but not methanogenesis from pyruvate. Although the specific roles of porE and porF have not been determined, these results suggest that PorEF play important roles in the anabolic POR in vivo even though they are not required for the dye-dependent activity.Abbreviations CODH/ACS Carbon monoxide dehydrogenase/acetyl-CoA synthase - POR Pyruvate oxidoreductase     

Metabolic flux and robustness analysis of glycerol metabolism in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The knowledge of the mechanism of flux distribution will benefit understanding cell physiology and regulation of metabolism. In this study, the measured fluxes obtained under steady-state conditions were used to estimate intracellular fluxes and identify the robustness of branch points of the anaerobic glycerol metabolism in Klebsiella pneumoniae for the production of 1,3-propanediol by metabolic flux analysis. The biomass concentration increased as NADH₂/NAD⁺ decreased at low initial concentration and inversed at high initial glycerol concentration. The flux distribution revealed that the branch points of glycerol and dihydroxyacetonephosphate were rigid to the environmental conditions. However, the pyruvate and acetyl coenzyme A metabolisms gave cells the flexibility to regulate the energy and intermediate fluxes under various environmental conditions. Additionly, it was found that the formation rate of ethanol and the ratio of pyruvate dehydrogenase to pyruvate formate lyase appeared visible fluctuations at high glycerol uptake rate.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-asparaginase in<Emphasis Type="Italic"> Enterobacter aerogenes</Emphasis> expressing<Emphasis Type="Italic"> Vitreoscilla</Emphasis> hemoglobin for efficient oxygen uptake

This study is the first utilizing Vitreoscilla hemoglobin in a heterologous bacterium, Enterobacter aerogenes, to determine the effect of such a highly efficient oxygen-uptake system on the production of l-asparaginase, an enzyme that has attracted considerable attention due to its anti-tumor activity. Here, we show that the Vitreoscilla hemoglobin expressing strain has from 10-fold to more than two orders of magnitude lower l-asparaginase activity than the wild type or the control without the Vitreoscilla hemoglobin gene under different aeration conditions. Aeration and agitation were also determining factors for enzyme production. The enzyme activity was reduced considerably under both full aerobic and anaerobic conditions, while the highest enzyme activity was determined in cultures under low aeration and low agitation. Also, the effect of different concentrations of glucose on enzyme production showed catabolic repression. Glucose at 1% caused almost total inhibition of enzyme activity, while at 0.1% it showed a slightly stimulatory effect on enzyme production, compared with glucose-free medium.     

End-product induced metabolic shifts in <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> ATCC 27405

When attempting to increase yields of desirable end-products during fermentation, there is the possibility that increased concentrations of one product redirects metabolism towards the synthesis of less desired products. Changes in growth, final end-product concentrations, and activities of enzymes involved in pyruvate catabolism and fermentative end-product formation were studied in Clostridium thermocellum in response to the addition of individual end-products (H₂, acetate, ethanol, formate, and lactate) to the growth medium. These were added to the growth medium at concentrations ten times greater than those found at the end of growth in cultures grown under carbon-limited conditions using cellobiose (1.1 g l⁻¹) as model soluble substrate. Although growth rate and final cell biomass decreased significantly with the addition of all end-products, addition of individual end-products had less pronounced effects on growth. Metabolic shifts, represented by changes in final end-product concentrations, were observed; H₂ and acetate yields increased in the presence of exogenous ethanol and lactate, while ethanol yields increased in the presence of exogenous hydrogen (H₂), acetate, and lactate. Late exponential phase enzyme activity data of enzymes involved in pyruvate catabolism and end-product formation revealed no changes in enzyme levels greater than 2-fold in response to the presence of any given end-product, with the exception of pyruvate:formate lyase (PFL), ferredoxin-dependent hydrogenase (Fd-H₂ase), and pyruvate:ferredoxin oxidoreductase (PFO): PFL and Fd-H₂ase activities increased 2-fold in the presence of ethanol, while PFO activity decreased by 57% in the presence of sodium formate. Changes in enzyme levels did not necessarily correlate with changes in final end-product yields, suggesting that changes in final end-product yields may be governed by thermodynamic considerations rather than levels of enzyme expressed under the conditions tested. We demonstrate that bacterial metabolism may be manipulated in order to selectively improve desired product yields.     

Ca<Superscript>2+</Superscript> Dependence and Inhibitory Effects of Trifluoperazine on Plasma Membrane ATPase of <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis>

Ca²⁺ enhanced the plasma membrane Ca²⁺-ATPase (PMCA) specific activities in wild-type strain 1227 and mutant strains 1278, 1286, and 1261 of Thermoactinomyces vulgaris. The Ca²⁺-ATPase specific activities showed marked increase with increasing concentrations of Ca²⁺ added in the form of CaCl₂ in the culture medium and reached the optimum values at 0.6 mM in strains 1227, 1278, and 1286 and at 0.7 mM in strain 1261 of T. vulgaris. Trifluoperazine, a specific blocker of calmodulin, when added in vivo at concentrations of 2 M and 8 M along with the respective optimal concentrations of Ca²⁺, decreased the PMCA-specific activities to a low level in a dose-dependent manner. The results of the present investigation suggest the presence of a Ca²⁺-dependent protein activator (CaDPA) in the microenvironment constituting this enzyme; and such Ca²⁺-modulated protein has been assigned to play an important role in the enhancement of PMCA levels in this aerobic, spore-forming, thermophilic actinomycete.