Synthesis and Biological Activity of Acinetobacter calcoaceticus IMV B-7241 Surfactants Depending on Monovalent Cations Content in Cultivation Medium

Microbial surfactants (biosurfactants) are multifunctional preparations due to a combination of physicochemical (reduction of surface and interfacial tension, emulsifying activity) and biological (antimicrobial and antiadhesive activity, the ability to destroy biofilms) properties. However, the disadvantage of biosurfactants synthesized as a complex of compounds is the possibility of changing the biological activity depending on the conditions of producer cultivation. Aim. To study the effect of potassium and sodium cations on the NADP+-dependent glutamate dehydrogenase activity of cell-free extract of Acinetobacter calcoaceticus ІМV B-7241 with subsequent appropriate modification of the nutrient medium composition and determination of antimicrobial and anti-adhesive activity of surfactant synthesized. Methods. A. calcoaceticus ІМV B-7241 strain was grown in media containing 2% of sunflower oil waste as a carbon source, as well as various concentrations of potassium and sodium chloride (basal – 1.0 g/l NaCl, medium #1 that did not contains NaCl, medium #2 in which the concentration of NaCl was 2.0 g/l, medium #3 in which the concentration of NaCl and KCl was 1.0 g/l each). The surfactants were extracted from the supernatant liquid culture with a modified Folch mixture. Antiadhesive activity and the degree of biofilms degradation were determined by spectrophotometric method, antimicrobial activity − by the indicator of the minimum inhibitory concentration (MIC). Activity of enzymes of surface-active aminolipids biosynthesis (NADP+-dependent glutamate dehydrogenase) and glycolipids (phosphoenolpyruvate (PEP) carboxylase, PEP-synthetase, PEP-carboxykinase, trehalose phosphate synthase) were analyzed in cell-free extracts obtained after the destruction of cells by ultrasound. Results. It was found that potassium and sodium cations in concentrations of 50 and 100 mM are inhibitors of NADP+-dependent glutamate dehydrogenase, and in lower concentrations (5–20 mM) – activators of this enzyme, as well as PEP-carboxykinase and PEP-synthetase. The increase in the biosurfactant concentration to 6.1−7.7 g/l during cultivation of A. calcoaceticus ІМV B-7241 in medium #1 and #3 was due to the predominant synthesis of glycolipids under such conditions, which was evidenced by the increase in 1.8−6.5 times in the activity of PEP-carboxylase, PEP-carboxykinase, PEP-synthetase and trehalose phosphate synthetase compared to the indicators on the basal medium. The concentration of surfactants synthesized in the basal medium was 3.6 g/l, but such surfactants were characterized by the highest antimicrobial and anti-adhesive activity. Their MIC against the test-cultures of studied bacteria (Pseudomonas sp. MI-2, Bacillus subtilis BT-2, Escherichia coli IEM-1, Staphylococcus aureus BMS-1, Enterobacter cloaceae C-8) and fungi (Candida albicans D-6, Rhizopus nigricans P1, Aspergillus niger P-3, Fusarium culmorum T-7) were 0.88−56 μg/ml and were by 2−3 orders of magnitude lower compared to established for surfactants synthesized in modified media #1–3. In the case of treatment of abiotic materials with surfactant solutions obtained on the basal medium, the adhesion of bacteria and fungi was on average 10–20% lower than after surface treatment by the surfactant synthesized in modified media. In the presence of 148−296 μg/ml of surfactants obtained in the basal medium, destruction of S. aureus BMS-1 and B. subtilis BT-2 biofilms was 45−66%, and C. albicans D-6 yeast – 39−44%. Under the action of similar concentrations of surfactants synthesized in modified media, the destruction of bacterial and yeast biofilms was lower: 6-52 and 20–46%, respectively. Conclusions. The obtained results are consistent with the data of our previous studies on the possibility of regulating the antimicrobial and antiadhesive activity of surfactants in the process of producer cultivation by changing the content of cations in the medium, which are inhibitors/activators of enzymes responsible for the synthesis of components of the surfactants complex, which have certain biological properties.

Atypical Glycolysis in Clostridium thermocellum

ABSTRACTCofactor specificities of glycolytic enzymes inClostridium thermocellumwere studied with cellobiose-grown cells from batch cultures. Intracellular glucose was phosphorylated by glucokinase using GTP rather than ATP. Although phosphofructokinase typically uses ATP as a phosphoryl donor, we found only pyrophosphate (PPi)-linked activity. Phosphoglycerate kinase used both GDP and ADP as phosphoryl acceptors. In agreement with the absence of a pyruvate kinase sequence in theC. thermocellumgenome, no activity of this enzyme could be detected. Also, the annotated pyruvate phosphate dikinase (ppdk) is not crucial for the generation of pyruvate from phosphoenolpyruvate (PEP), as deletion of theppdkgene did not substantially change cellobiose fermentation. Instead pyruvate formation is likely to proceed via a malate shunt with GDP-linked PEP carboxykinase, NADH-linked malate dehydrogenase, and NADP-linked malic enzyme. High activities of these enzymes were detected in extracts of cellobiose-grown cells. Our results thus show that GTP is consumed while both GTP and ATP are produced in glycolysis ofC. thermocellum. The requirement for PPiin this pathway can be satisfied only to a small extent by biosynthetic reactions, in contrast to what is generally assumed for a PPi-dependent glycolysis in anaerobic heterotrophs. Metabolic network analysis showed that most of the required PPimust be generated via ATP or GTP hydrolysis exclusive of that which happens during biosynthesis. Experimental proof for the necessity of an alternative mechanism of PPigeneration was obtained by studying the glycolysis in washed-cell suspensions in which biosynthesis was absent. Under these conditions, cells still fermented cellobiose to ethanol.

Pyruvate Is Synthesized by Two Pathways in Pea Bacteroids with Different Efficiencies for Nitrogen Fixation

ABSTRACT Nitrogen fixation in legume bacteroids is energized by the metabolism of dicarboxylic acids, which requires their oxidation to both oxaloacetate and pyruvate. In alfalfa bacteroids, production of pyruvate requires NAD+ malic enzyme (Dme) but not NADP+ malic enzyme (Tme). However, we show that Rhizobium leguminosarum has two pathways for pyruvate formation from dicarboxylates catalyzed by Dme and by the combined activities of phosphoenolpyruvate (PEP) carboxykinase (PckA) and pyruvate kinase (PykA). Both pathways enable N2 fixation, but the PckA/PykA pathway supports N2 fixation at only 60% of that for Dme. Double mutants of dme and pckA/pykA did not fix N2. Furthermore, dme pykA double mutants did not grow on dicarboxylates, showing that they are the only pathways for the production of pyruvate from dicarboxylates normally expressed. PckA is not expressed in alfalfa bacteroids, resulting in an obligate requirement for Dme for pyruvate formation and N2 fixation. When PckA was expressed from a constitutive nptII promoter in alfalfa dme bacteroids, acetylene was reduced at 30% of the wild-type rate, although this level was insufficient to prevent nitrogen starvation. Dme has N-terminal, malic enzyme (Me), and C-terminal phosphotransacetylase (Pta) domains. Deleting the Pta domain increased the peak acetylene reduction rate in 4-week-old pea plants to 140 to 150% of the wild-type rate, and this was accompanied by increased nodule mass. Plants infected with Pta deletion mutants did not have increased dry weight, demonstrating that there is not a sustained change in nitrogen fixation throughout growth. This indicates a complex relationship between pyruvate synthesis in bacteroids, nitrogen fixation, and plant growth.

Cyclic AMP-Dependent Catabolite Repression Is the Dominant Control Mechanism of Metabolic Fluxes under Glucose Limitation in Escherichia coli

ABSTRACT Although a whole arsenal of mechanisms are potentially involved in metabolic regulation, it is largely uncertain when, under which conditions, and to which extent a particular mechanism actually controls network fluxes and thus cellular physiology. Based on 13C flux analysis of Escherichia coli mutants, we elucidated the relevance of global transcriptional regulation by ArcA, ArcB, Cra, CreB, CreC, Crp, Cya, Fnr, Hns, Mlc, OmpR, and UspA on aerobic glucose catabolism in glucose-limited chemostat cultures at a growth rate of 0.1 h−1. The by far most relevant control mechanism was cyclic AMP (cAMP)-dependent catabolite repression as the inducer of the phosphoenolpyruvate (PEP)-glyoxylate cycle and thus low tricarboxylic acid cycle fluxes. While all other mutants and the reference E. coli strain exhibited high glyoxylate shunt and PEP carboxykinase fluxes, and thus high PEP-glyoxylate cycle flux, this cycle was essentially abolished in both the Crp and Cya mutants, which lack the cAMP-cAMP receptor protein complex. Most other mutations were phenotypically silent, and only the Cra and Hns mutants exhibited slightly altered flux distributions through PEP carboxykinase and the tricarboxylic acid cycle, respectively. The Cra effect on PEP carboxykinase was probably the consequence of a specific control mechanism, while the Hns effect appears to be unspecific. For central metabolism, the available data thus suggest that a single transcriptional regulation process exerts the dominant control under a given condition and this control is highly specific for a single pathway or cycle within the network.

Genome-Based Metabolic Engineering of Mannheimia succiniciproducens for Succinic Acid Production

ABSTRACT Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO2-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.