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Archives of Microbiology (v.171, #1)
The anaerobic degradation of l-serine and l-threonine in enterobacteria: networks of pathways and regulatory signals by Gary Sawers (pp. 1-5).
The mechanisms controlling the biosynthesis and degradation of l-serine and l-threonine are remarkably complex. Their metabolism forms a network of pathways linking several amino acids, central primary metabolites such as pyruvate, oxaloacetate and 3-phosphoglycerate, and C1 metabolism. Studies on the degradation of these amino acids in Escherichia coli have revealed the involvement of fascinating enzymes that utilise quite diverse catalytic mechanisms. Moreover, it is emerging that both environmental and metabolic signals have a major impact in controlling enzyme synthesis. This is exemplified by the anaerobically regulated tdc operon, which encodes a metabolic pathway for the degradation of serine and threonine. Studies on this pathway are beginning to provide insights into how an organism adapts its genetic makeup to meet the physiological demands of the cell.
Keywords: Key wordsl-Serine; l-threonine; Anaerobiosis; Pyridoxal 5′-phosphate enzymes; Iron-sulfur enzymes; Glycyl radical enzymes; Metabolism
Activation of the H+-ATPase in the plasma membrane of cells of Saccharomyces cerevisiae grown under mild copper stress by Alexandra R. Fernandes; Francisco P. Peixoto; I. Sá-Correia (pp. 6-12).
Cells of Saccharomyces cerevisiae exibited a more active plasma membrane H+-ATPase during growth in media supplemented with CuSO4 concentrations equal to or below 1 mM than did cells cultivated in the absence of copper stress. Maximal specific activities were found with 0.5 mM CuSO4. ATPase activity declined when cells were grown with higher concentrations up to 1.5 mM (the maximal concentration that allowed growth), probably due to severe disorganization of plasma membrane. Cu2+-induced maximal activation was reflected in an increase of V max (approximately threefold) and in the slight decrease of the K m for MgATP (from 0.93 ± 0.13 to 0.65 ± 0.16 mM). The expression of the gene encoding the essential plasma membrane ATPase (PMA1) was reduced with a dose-dependent pattern in cells grown with inhibitory concentrations of copper, while the weakly expressed PMA2 gene promoter was moderately more efficient in cells cultivated under mild copper stress (1.5-fold maximal activation). ATPase was activated by copper despite the slightly lower content of ATPase protein in the plasma membrane of Cu2+-grown cells and the powerful inhibitory effect of Cu2+ in vitro.
Keywords: Key words Plasma membrane H+-ATPase; Saccharomyces cerevisiae; Copper stress; PMA1; PMA2; Gene expression
Effect of high sugar concentration on nitrogenase activity of Acetobacter diazotrophicus by V. M. Reis; Johanna Döbereiner (pp. 13-18).
Acetobacter diazotrophicus is a nitrogen-fixing bacterium that grows inside sugar cane plant tissue where the sucrose concentration is approximately 10%. The influence of high sugar content on nitrogenase was measured in the presence of oxygen and of nitrogen added in the form of ammonium and amino acids. In all parameters analyzed, 10% sucrose protected nitrogenase against inhibition by oxygen, ammonium, some amino acids, and also to some extent by salt stress. The oxygen concentration at which inhibition occurred increased from 2 kPa in 1% glucose or gluconic acid, to 4 kPa (0.4 atm) in 10% sucrose. Nitrogenase activity was partially inhibited by increased ammonium levels (2.0, 5.0, and 10.0 mM) in the presence of 1% sucrose, but the cells maintained their nitrogenase activity at 10% sucrose. This could be explained by the slow ammonium assimilation by the cells in the presence of high sucrose concentrations, i.e., independent of its concentration between 2 and 10 mM, the assimilation of ammonium was reduced to one-third in cells grown with 10% sucrose. Some amino acids were also tested in the presence of 1 and 10% sucrose. Cells grown in 1% sucrose had their nitrogenase activity reduced by 50–98% in the presence of glutamic acid, glutamine, alanine, asparagine, or threonine, whereas with 10% sucrose, nitrogenase activity was increased by glutamic acid and was reduced by only 61–73% by the other amino acids. The effect of NaCl concentrations (0.0, 0.25, 0.5, 0.75, or 1.0%) was also studied at the two concentrations of sucrose. Nitrogenase activity and growth of A. diazotrophicus, which was visualized by the pellicle formation in semi-solid medium, showed sensitivity even to low NaCl concentrations, which was somewhat relieved at the higher sucrose level. These observations indicate different osmotolerance mechanisms for sucrose and salt.
Keywords: Key wordsAcetobacter diazotrophicus; Ammonium; Nitrogenase activity; Oxygen; Osmotolerance
Bacillus arsenicoselenatis, sp. nov., and Bacillus selenitireducens, sp. nov.: two haloalkaliphiles from Mono Lake, California that respire oxyanions of selenium and arsenic by Jodi Switzer Blum; A. Burns Bindi; J. Buzzelli; John F. Stolz; R. S. Oremland (pp. 19-30).
Two gram-positive anaerobic bacteria (strains E1H and MLS10) were isolated from the anoxic muds of Mono Lake, California, an alkaline, hypersaline, arsenic-rich water body. Both grew by dissimilatory reduction of As(V) to As(III) with the concomitant oxidation of lactate to acetate plus CO2. Bacillus arsenicoselenatis (strain E1H) is a spore-forming rod that also grew by dissimilatory reduction of Se(VI) to Se(IV). Bacillus selenitireducens (strain MLS10) is a short, non-spore-forming rod that grew by dissimilatory reduction of Se(IV) to Se(0). When the two isolates were cocultured, a complete reduction of Se(VI) to Se(0) was achieved. Both isolates are alkaliphiles and had optimal specific growth rates in the pH range of 8.5–10. Strain E1H had a salinity optimum at 60 g l–1 NaCl, while strain MLS10 had optimal growth at lower salinities (24–60 g l–1 NaCl). Both strains have limited abilities to grow with electron donors and acceptors other than those given above. Strain MLS10 demonstrated weak growth as a microaerophile and was also capable of fermentative growth on glucose, while strain E1H is a strict anaerobe. Comparative 16S rRNA gene sequence analysis placed the two isolates with other Bacillus spp. in the low G+C gram-positive group of bacteria.
Studies of the production and characterization of laccase activity in the basidiomycete Coriolopsis gallica, an efficient decolorizer of alkaline effluents by A. M. Calvo; José L. Copa-Patiño; Oriele Alonso; A. E. González (pp. 31-36).
The basidiomycete Coriolopsis gallica decolorizes alkaline paper effluents efficiently. In this work, we found that C. gallica produces laccase during this decolorization process. This enzymatic activity was produced in all media studied; however, the highest enzymatic activity was obtained in a medium containing paper effluent, where laccase was detected on the 2nd day of the experiment. The laccase activity of C. gallica was purified and characterized. The amino-terminal sequence of this protein showed the highest similarity with the laccase I of the basidiomycete PM1 and with Coriolus hirsutus laccase.
Keywords: Key words Laccase; Coriolopsis gallica; Decolorization; Alkali lignin
Purification and characterization of eugenol dehydrogenase from Pseudomonas fluorescens E118 by Hirotaka Furukawa; Marco Wieser; Hiroshi Morita; Tsuyoshi Sugio; T. Nagasawa (pp. 37-43).
Pseudomonas fluorescens E118 was isolated from soil as an effective eugenol-degrading organism by a screening using eugenol as enrichment substrate. The first enzyme involved in the degradation of eugenol in this organism, eugenol dehydrogenase, was purified after induction by eugenol, and the purity of the enzyme was shown by SDS-PAGE and gel-permeation HLPC. The enzyme is a heterodimer that consists of a 10-kDa cytochrome c and a 58-kDa subunit. The larger subunit presumably contains flavin, suggesting a flavocytochrome c structure and an electron transfer via flavin and cytochrome c during dehydrogenation. The activity of the purified enzyme depended on the addition of a final electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol, cytochrome c, or potassium ferricyanide. The enzyme catalyzed the dehydrogenation of three different 4-hydroxybenzylic structures including the conversion of eugenol to coniferyl alcohol, 4-alkylphenols to 1-(4-hydroxyphenyl)alcohols, and 4-hydroxybenzylalcohols to the corresponding aldehydes. The catalytic and structural similarity between this enzyme and a Penicillium vanillyl-alcohol oxidase and 4-alkylphenol methylhydroxylases from several Pseudomonas species is discussed.
Keywords: Key words Dehydrogenase; Eugenol; Purification; Flavocytochrome c; Pseudomonas fluorescens E118; Substrate specificity; 4-Alkylphenol; 4-Hydroxybenzyl alcohol; Electron acceptor
Oxidation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes eutrophus A5 by Lloyd J. Nadeau; Gary S. Sayler; J. C. Spain (pp. 44-49).
Previous studies demonstrated that Alcaligenes eutrophus A5 transforms 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) to 4-chlorobenzoate via a meta-ring fission product. The initial reactions could be catalyzed by either monooxygenase or dioxygenase enzymes. In the present study, a transient intermediate that accumulated during the transformation of DDT by the biphenyl-grown cells was identified as 1,1,1-trichloro-2-(4-chlorophenyl-2,3-dihydro-4,6-cyclohexadiene)-2-(4′-chlorophenyl)ethane (DDT-2,3-dihydrodiol) on the basis of mass spectral analysis after n-butylboronic acid derivatization. The dihydrodiol undergoes a characteristic acid-catalyzed dehydration to produce phenols. 1H-NMR indicated a cis-relative stereochemistry. The results indicate that the biphenyl dioxygenase from A. eutrophus A5 catalyzes the dihydroxylation of DDT at the unsubstituted carbons on the aromatic ring to produce DDT-2,3-dihydrodiol.
Keywords: Key words DDT dioxygenation; Alcaligenes eutrophus; A5; Dioxygenase; DDT-2; 3-dihydrodiol; Polychlorinated; aromatic compound
Inhibition of colicin synthesis by the antibiotic globomycin by Danièle Cavard (pp. 50-58).
The antibiotic globomycin, an inhibitor of LspA (the lipoprotein signal peptidase), inhibited synthesis of colicin by Escherichia coli cells grown in rich medium. This inhibition was stronger in cells with mutation(s) within either the colicin operon, which is located on a plasmid, or the host chromosome. This phenotype was called Gbc (globomycin blocks colicin synthesis). The Gbc phenotype was affected by growth conditions since it was partially or totally suppressed in cells subjected to high temperatures, treated with sodium azide, or grown in minimal medium. The Gbc phenotype observed with colicin-A-producing cells was more severe in strains carrying plasmids with a deletion within caa (the first gene of the colicin A operon), which encodes colicin A, than in cells with the wild-type caa gene. The Gbc phenotype was alleviated by a null mutation in the degP gene encoding the DegP/HtrA protease, abolished by a null mutation in the lpp gene encoding the murein-lipoprotein, and enhanced by a mutation in the pldA gene encoding the outer membrane phospholipase A. Transcription of the colicin A operon was blocked in cells exhibiting the Gbc phenotype as evidenced by rifampicin treatment of induced cells. This phenotype suggests that either a lipoprotein or a protein involved in lipoprotein metabolism might be involved in the regulation of the expression of the colicin operons and that the colicin A structural gene might play a role in the regulation of transcription of the colicin A operon.
Keywords: Key wordsEscherichia coli; Colicin; Globomycin; Lipoprotein; Transcription
Isolation and characterization of enterocin EJ97, a bacteriocin produced by Enterococcus faecalis EJ97 by A. Gálvez; E. Valdivia; Hikmate Abriouel; Emilio Camafeita; Enrique Mendez; Manuel Martínez-Bueno; Mercedes Maqueda (pp. 59-65).
The bacteriocinogenic strain of Enterococcus faecalis EJ97 has been isolated from municipal waste water. It produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,340 Da) that is very stable under mild heat conditions and is sensitive to proteolytic enzymes. The amino acid sequence of the first 18 N-terminal residues of enterocin EJ97 indicates that it is different from other known protein sequences. Enterocin EJ97 is active on several gram-positive bacteria including enterococci, several species of Bacillus, Listeria, and Staphylococcus aureus. The producer strain is immune to bacteriocin. Enterocin EJ97 has a concentration-dependent bactericidal and bacteriolytic effect on E. faecalis S-47.
Keywords: Key words Bacteriocin; Enterococcus; Listeria; Cationic peptide
Biosynthesis of cobalamin in Salmonella typhimurium: transformation of riboflavin into the 5,6-dimethylbenzimidazole moiety by Bettina Keck; Michael Munder; P. Renz (pp. 66-68).
In order to elucidate the biosynthesis of the base moiety of cobalamin in Salmonella typhimurium LT2, this organism was grown in the presence of [1′-14C]riboflavin. The vitamin B12 isolated was 14C-labeled. It was shown by chemical degradation that the 14C-label was exclusively localized in carbon atom 2 of the 5,6-dimethylbenzimidazole moiety. This demonstrated the precursor function of riboflavin in the biosynthesis of 5,6-dimethylbenzimidazole in S. typhimurium.
Keywords: Key words 5; 6-Dimethylbenzimidazole; Riboflavin; Salmonella typhimurium; Vitamin B12
Effect of oxidative stress on the biosynthesis of 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate and isoprenoids by several bacterial strains by D. Ostrovsky; G. Diomina; E. Lysak; E. Matveeva; O. Ogrel; S. Trutko (pp. 69-72).
In this study, the gram-negative bacteria Xanthomonas campestris, Xanthomonas maltophilia, and Pseudomonas putida, facultative parasites of plants and animals, were shown to accumulate 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate (MEC) in response to benzyl-viologen-induced oxidative stress. Corynebacterium ammoniagenes mutants capable of accumulating MEC in the absence of an exogenous oxidative stress inducer were obtained. Isoprenoid synthesis and MEC synthesis in these and other bacteria were shown to be alternative processes, while biosynthesis of brominated polyene xanthomonadin (an antioxidant pigment of X. campestris) increased concomitantly with the accumulation of MEC.
Keywords: Key words 2-C-methyl-d-erythritol; Xanthomonas; Pseudomonas; Corynebacterium; Isoprenoid; Phosphate