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Archives of Microbiology (v.170, #2)
Energy conservation in the decarboxylation of dicarboxylic acids by fermenting bacteria by Peter Dimroth; B. Schink (pp. 69-77).
Decarboxylation of dicarboxylic acids (oxalate, malonate, succinate, glutarate, and malate) can serve as the sole energy source for the growth of fermenting bacteria. Since the free energy change of a decarboxylation reaction is small (around –20 kJ per mol) and equivalent to only approximately one-third of the energy required for ATP synthesis from ADP and phosphate under physiological conditions, the decarboxylation energy cannot be conserved by substrate-level phosphorylation. It is either converted (in malonate, succinate, and glutarate fermentation) by membrane-bound primary decarboxylase sodium ion pumps into an electrochemical gradient of sodium ions across the membrane; or, alternatively, an electrochemical proton gradient can be established by the combined action of a soluble decarboxylase with a dicarboxylate/monocarboxylate antiporter (in oxalate and malate fermentation). The thus generated electrochemical Na+ or H+ gradients are then exploited for ATP synthesis by Na+- or H+-coupled F1F0 ATP synthases. This new type of energy conservation has been termed decarboxylation phosphorylation and is responsible entirely for ATP synthesis in several anaerobic bacteria.
Keywords: Key words ATP synthase; Decarboxylation; Electrogenic substrate/product antiporter; Sodium ion; pump; Malo-lactic fermentation; Oxalate; Malonate; Succinate; Glutarate
Purification and characterization of laccase II of Aspergillus nidulans by Mario Scherer; R. Fischer (pp. 78-84).
Sexual development in Aspergillus nidulans is a morphogenetic differentiation process triggered by internal and environmental signals. As a first step in analyzing the developmental pathway at the molecular level, laccase II (EC 1.10.3.2), which is specifically expressed in early stages of fruitbodies, was isolated. The enzyme was purified to apparent homogeneity from a mutant strain (SMS1) in which the sexual cycle dominates and the number of cleistothecia is increased tenfold. Laccase II was enriched 560-fold to a specific activity of 892 U (mg protein)–1. The apparent molecular mass was determined to be 80 kDa under denaturing conditions and to be 100–120 kDa under native conditions. The internal peptide sequences gained from the protein will allow the isolation of the corresponding gene as a first step in determining the key regulators of sexual development.
Keywords: Key wordsAspergillus nidulans; Filamentous fungi; Sexual development; Cleistothecia; Laccase
Isolation and characterization of a bacterium possessing a novel aldoxime-dehydration activity and nitrile-degrading enzymes by Yasuo Kato; Ryoko Ooi; Y. Asano (pp. 85-90).
A bacterial strain capable of utilizing E-pyridine-3-aldoxime as a nitrogen source was isolated from soil after a 4-month acclimation period and was identified as Rhodococcus sp. The strain contained a novel aldoxime dehydration activity that catalyzed a stoichiometric dehydration of E-pyridine-3-aldoxime to form 3-cyanopyridine. The enzyme activity was induced by various aldoximes and nitriles. The strain metabolized the aldoxime as follows: E-pyridine-3-aldoxime was dehydrated to form 3-cyanopyridine, which was converted to nicotinamide by a nitrile hydratase, and the nicotinamide was successively hydrolyzed to nicotinic acid by an amidase.
Keywords: Key words Aldoxime dehydration; Synthesis; Aldoxime; Nitrile; Rhodococcus sp.
Purification and characterization of a dextranase from Sporothrix schenckii by W. N. Arnold; Tan Binh P. Nguyen; Larry C. Mann (pp. 91-98).
A dextranase (EC 3.2.1.11) was purified and characterized from the IP-29 strain of Sporothrix schenckii, a dimorphic pathogenic fungus. Growing cells secreted the enzyme into a standard culture medium (20 °C) that supports the mycelial phase. Soluble bacterial dextrans substituted for glucose as substrate with a small decrease in cellular yield but a tenfold increase in the production of dextranase. This enzyme is a monomeric protein with a molecular mass of 79 kDa, a pH optimum of 5.0, and an action pattern against a soluble 170-kDa bacterial dextran that leads to a final mixture of glucose (38%), isomaltose (38%), and branched oligosaccharides (24%). In the presence of 200 mM sodium acetate buffer (pH 5.0), the K m for soluble dextran was 0.067 ± 0.003% (w/v). Salts of Hg2+, (UO2)2+, Pb2+, Cu2+, and Zn2+ inhibited by affecting both V max and K m. The enzyme was most stable between pH values of 4.50 and 4.75, where the half-life at 55 °C was 18 min and the energy of activation for heat denaturation was 99 kcal/mol. S. schenckii dextranase catalyzed the degradation of cross-linked dextran chains in Sephadex G-50 to G-200, and the latter was a good substrate for cell growth at 20 °C. Highly cross-linked grades (i.e., G-10 and G-25) were refractory to hydrolysis. Most strains of S. schenckii from Europe and North America tested positive for dextranase when grown at 20 °C. All of these isolates grew on glucose at 35 °C, a condition that is typically associated with the yeast phase, but they did not express dextranase and were incapable of using dextran as a carbon source at the higher temperature.
Keywords: Key words Dextran; Dextranase; Sporothrix schenckii; Pathogenic fungus; Dimorphism
Physiological and genetic characterisation of osmosensitive mutants of Saccharomyes cerevisiae by Adrian R. N. E. Brüning; Jürgen Bauer; Bernhard Krems; Karl-Dieter Entian; B. A. Prior (pp. 99-105).
The screening of 20,000 Saccharomyces cerevisiae random mutants to identify genes involved in the osmotic stress response yielded 14 mutants whose growth was poor in the presence of elevated concentrations of NaCl and glucose. Most of the mutant strains were more sensitive to NaCl than to glucose at the equivalent water activity (aw) and were classified as salt-sensitive rather than osmosensitive. These mutants fell into 11 genetic complementation groups and were designated osr1–osr11 (osmotic stress response). All mutations were recessive and showed a clear 2+ : 2– segregation of the salt-stress phenotype upon tetrad analysis when crossed to a wild-type strain. The complementation groups osr1, osr5 and osr11 were allelic to the genes PBS2, GPD1 and KAR3, respectively. Whereas intracellular and extracellular levels of glycerol increased in the wild-type strains when exposed to NaCl, all mutants demonstrated some increase in extracellular glycerol production upon salt stress, but a number of the mutants showed little or no increase in intracellular glycerol concentrations. The mutants had levels of glycerol-3-phosphate dehydrogenase, an enzyme induced by osmotic stress, either lower than or similar to those of the parent wild-type strain in the absence of osmotic stress. In the presence of NaCl, the increase in glycerol-3-phosphate dehydrogenase activity in the mutants did not match that of the parent wild-type strain. None of the mutants had defective ATPases or were sensitive to heat stress. It is evident from this study and from others that a wide spectrum of genes is involved in the osmotic stress response in S. cerevisiae.
Keywords: Key words Osmosis; Yeast; Mutant; Glycerol; Solute; Complementation group
Purification and characterization of the NADH:ferredoxinBPH oxidoreductase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400 by Richard M. Broadus; J. D. Haddock (pp. 106-112).
NADH:ferredoxinBPH oxidoreductase (reductaseBPH) of biphenyl 2,3-dioxygenase was purified over 47-fold to homogeneity with a yield of 41% from cell extract of Pseudomonas sp. strain LB400. ReductaseBPH transfers reducing equivalents from NADH to the catalytic oxygenase component (ISPBPH) via a ferredoxin (ferredoxinBPH) during the oxidation of biphenyl to cis-biphenyl 2,3-dihydrodiol. ReductaseBPH was a monomer with a molecular weight of 43,600 as determined by electrophoresis under denaturing conditions. Gel filtration column chromatography gave a molecular weight of 41,500 for native reductaseBPH. The absorbance spectrum of the protein in its oxidized state had maxima at 271 nm, 376 nm and 448 nm with shoulders at 422 nm and 476 nm. The peak around 448 nm was partially bleached upon reduction with NADH under anoxic conditions. ReductaseBPH contained 0.89 mol FAD/mol protein. ReductaseBPH was required for oxidation of biphenyl to cis-biphenyl 2,3-dihydrodiol by ISPBPH and ferredoxinBPH. Potassium ferricyanide, 2,6-dichlorophenolindophenol (DCPIP), nitrobluetetrazolium and cytochrome c served as artificial electron acceptors. Reduction of cytochrome c was dependent upon the presence of ferredoxinBPH. The fastest rate of DCPIP reduction occurred at pH 7.2 and 32° C. The apparent K m for NADH and NADPH in the DCPIP assay were 58 μM and 156 μM, respectively. V max was 3,120 U mg–1 for NADH and 1,140 U mg–1 for NADPH. NADH is most likely the physiological electron donor for oxidation of biphenyl and polychlorinated biphenyls.
Keywords: Key words Oxidoreductase; Flavoprotein; Dioxygenase; Biphenyl; Polychlorinated biphenyl; Electron transport; Pseudomonas
Candida albicans HEX1 gene, a reporter of gene expression in Saccharomyces cerevisiae by K. Niimi; Maxwell G. Shepherd; Richard D. Cannon (pp. 113-119).
The Candida albicans HEX1 gene was examined as a reporter of gene expression in Saccharomyces cerevisiae. The galactose-inducible S. cerevisiae GAL1-GAL10 promoter was inserted upstream of the C. albicans HEX1 gene, which encodes N-acetylglucosaminidase. The gene was introduced into S. cerevisiae AH22, which has no background N-acetylglucosaminidase activity. Expression of HEX1 in transformed cells was induced significantly by galactose and was repressed by glucose. The HEX1 gene product was functional in S. cerevisiae cells and was targeted to the periplasm. Both untransformed S. cerevisiae cells and cells expressing HEX1 had similar growth curves and cell morphology indicating that expression of N-acetylglucosaminidase was not toxic to the host strain. These results demonstrate that the HEX1 gene can be a useful reporter of gene expression in S. cerevisiae.
Keywords: Key wordsCandida albicans; Saccharomyces; cerevisiae; N-acetylglucosaminidase; HEX1; Reporter gene
Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica by J. Heider; Matthias Boll; Klaus Breese; Sabine Breinig; Christa Ebenau-Jehle; Ulrich Feil; Nasser Gad’on; Diana Laempe; Birgitta Leuthner; M. El-Said Mohamed; Sabine Schneider; Gerhard Burchhardt; Georg Fuchs (pp. 120-131).
Differential induction of enzymes involved in anaerobic metabolism of aromatic substrates was studied in the denitrifying bacterium Thauera aromatica. This metabolism is divided into (1) peripheral reactions transforming the aromatic growth substrates to the common intermediate benzoyl-CoA, (2) the central benzoyl-CoA pathway comprising ring-reduction of benzoyl-CoA and subsequent β-oxidation to 3-hydroxypimelyl-CoA, and (3) the pathway of β-oxidation of 3-hydroxypimelyl-CoA to three acetyl-CoA and CO2. Regulation was studied by three methods. 1. Determination of protein patterns of cells grown on different substrates. This revealed several strongly substrate-induced polypeptides that were missing in cells grown on benzoate or other intermediates of the respective metabolic pathways. 2. Measurement of activities of known enzymes involved in this metabolism in cells grown on different substrates. The enzyme pattern found is consistent with the regulatory pattern deduced from simultaneous adaptation of cells to utilisation of other aromatic substrates. 3. Immunological detection of catabolic enzymes in cells grown on different substrates. Benzoate-CoA ligase and 4-hydroxybenzoate-CoA ligase were detected only in cells yielding the respective enzyme activity. However, presence of the subunits of benzoyl-CoA reductase and 4-hydroxybenzoyl-CoA reductase was also recorded in some cell batches lacking enzyme activity. This possibly indicates an additional level of regulation on protein level for these two reductases.
Keywords: Key words Anaerobic aromatic metabolism; Benzoyl-CoA reductase; Phenylphosphate carboxylase; 4-Hydroxybenzoyl-CoA reductase; 2-Aminobenzoate; Phenylalanine; Phenylacetyl-CoA; Phenylglyoxylate; Toluene; CoA ligase
Metabolism of 2-aminophenol by Pseudomonas sp. AP-3: modified meta-cleavage pathway by Shinji Takenaka; Shuichiro Murakami; R. Shinke; K. Aoki (pp. 132-137).
A novel pathway for 2-aminophenol metabolism by Pseudomonas sp. AP-3 is proposed. The proposed pathway is similar to that known for meta-cleavage of catechol except that one of the hydroxyl groups on the metabolites is replaced by an amino group. During the degradation of 2-aminophenol, 2-amino-2,4-pentadienoic acid is the last metabolite containing an amino group. We, therefore, propose a modified meta-cleavage pathway for the 2-aminophenol metabolism.
Keywords: Key words Modified meta-cleavage pathway; 2-Aminophenol degradation; 2-Amino-2; 4-pentadienoic acid; 2-Oxopent-4-enoic acid; Pyruvic acid
Confirmation that Thiobacillus halophilus and Thiobacillus hydrothermalis are distinct species within the γ-subclass of the Proteobacteria by D. P. Kelly; Erko Stackebrandt; Jutta Burghardt; Ann P. Wood (pp. 138-140).
Thiobacillus halophilus and Thiobacillus hydrothermalis share 98.7% similarity in 16S rRNA sequence, possess similar gross DNA composition (64.2 and 67.4 mol% G+C values, respectively), and have similar physiological properties. While this might have indicated that they were strains of a single species, DNA-DNA hybridization between the type strains of the two species showed only 59% hybridization, indicating the organisms to be different at the species level. Thiobacillus neapolitanus is the phylogenetically nearest neighbour of T. halophilus and T. hydrothermalis (91.6–92.1% similarity in 16S rRNA sequence) and is the only other Thiobacillus in the γ-subclass of the Proteobacteria that can be regarded as exclusively related to these two species. The 16S rRNA gene sequences of these three species are so different from those of the other thiobacilli in the γ-subclass that they justify recognition as a distinct phyletic group. Their comparative properties are summarized.
Keywords: Key wordsThiobacillus taxonomy; Thiobacillus halophilus; Thiobacillus hydrothermalis; DNA; hybridization; mol% G+C; 16S rRNA sequences