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Archives of Microbiology (v.167, #1)
Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3 by J. F. Stolz; Theresa Gugliuzza; Jodi Switzer Blum; Ronald Oremland; Francisco Martínez Murillo (pp. 1-5).
The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b 554 (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b 556 (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth.
Respiratory stimulation and generation of superoxide radicals in Pseudomonas aeruginosa by fungal naphthoquinones by H. Haraguchi; Kazumi Yokoyama; Shingo Oike; Masakazu Ito; Hiroshi Nozaki (pp. 6-10).
The mechanism of action of antimicrobial naphthoquinones from the fungus Fusarium was studied by using Pseudomonas aeruginosa. Bostricoidin, methyl ether fusarubin, and fusarubin stimulated the oxygen consumption of bacterial cells and induced cyanide-insensitive oxygen consumption. These activities of the tested compounds were also observed in bacterial membrane preparations in a dose-dependent manner. Naphthoquinones stimulated the generation of superoxide anion and hydrogen peroxide. The naphthoquinone effectively acted as the electron acceptors for bacterial diaphorase, which could explain the antibacterial activity of Fusarium naphthoquinones since electron acceptors lead to the stimulation of respiratory activity and the generation of oxygen radical species.
Keywords: Key words Fungal naphthoquinones; Respiratory; burst; Superoxide anion; Electron acceptor; Antibacterial activity
Chromatium glycolicum sp. nov., a moderately halophilic purple sulfur bacterium that uses glycolate as substrate by P. Caumette; J. F. Imhoff; J. Süling; R. Matheron (pp. 11-18).
From the microbial mats that develop in Solar Lake, a new purple sulfur bacterium was isolated. This strain (Chromatium strain SL 3201) was morphologically similar to Chromatium gracile and Chromatium minutissimum. Chromatium SL 3201 was found to be a moderate halophile with a growth range between 2 and 20% NaCl (optimum 4–5% NaCl) and was able to grow photo-organotrophically using glycolate and glycerol. It is the first described phototrophic sulfur bacterium able to use glycolate. According to NaCl requirements and utilization of organic compounds, the strain is not related to any known species of the genus Chromatium. On the basis of its 16S rRNA gene sequence, it clusters with other Chromatium species and is most similar to Chromatium salexigens and Chr. gracile, but it is sufficiently separated to be considered as a new species of the genus. It is, therefore, described as Chromatium glycolicum sp. nov.
Keywords: Key wordsChromatium glycolicum; Phototrophic; bacteria; Halophilic bacteria; Hypersaline ecosystems; Microbial mats
Pathways of autotrophic CO2 fixation and of dissimilatory nitrate reduction to N2O in Ferroglobus placidus by J. A. Vorholt; Doris Hafenbradl; Karl O. Stetter; Rudolf K. Thauer (pp. 19-23).
The strictly anaerobic Archaeon Ferroglobus placidus was grown chemolithoautotrophically on H2 and nitrate and analyzed for enzymes and coenzymes possibly involved in autotrophic CO2 fixation. The following enzymes were found [values in parentheses = μmol min–1 (mg protein)–1]: formylmethanofuran dehydrogenase (0.2), formylmethanofuran:tetrahydromethanopterin formyltransferase (0.6), methenyltetrahydromethanopterin cyclohydrolase (10), F420-dependent methylenetetrahydromethanopterin dehydrogenase (1.5), F420-dependent methylenetetrahydromethanopterin reductase (0.4), and carbon monoxide dehydrogenase (0.1). The cells contained coenzyme F420 (0.4 nmol/mg protein), tetrahydromethanopterin (0.9 nmol/ mg protein), and cytochrome b (4 nmol/mg membrane protein). From the enzyme and coenzyme composition of the cells, we deduced that autotrophic CO2 fixation in F. placidus proceeds via the carbon monoxide dehydrogenase pathway as in autotrophically growing Archaeoglobus and Methanoarchaea species. Evidence is also presented that cell extracts of F. placidus catalyze the reduction of two molecules of nitrite to 1 N2O with NO as intermediate (0.1 μmol N2O formed per min and mg protein), showing that – at least in principle –F. placidus has a denitrifying capacity.
Keywords: Key words Autotrophic CO2 fixation; Dissimilatory; nitrate reduction; Archaeoglobus species; Methanogenic Archaea; Methanofuran; Tetrahydromethanopterin; Coenzyme F420; Cytochromes
Haem O and a putative cytochrome bo in a mutant of Bacillus cereus impaired in the synthesis of haem A by I. P. Del Arenal; M. L. Contreras; B. B. Svlateorova; P. Rangel; F. Lledías; J. R. Dávila; J. E. Escamilla (pp. 24-31).
In a spontaneous mutant (PYM1) of Bacillus cereus impaired in the synthesis of haem A, no haem-A-containing cytochromes were detected spectroscopically. The haem A deficiency was compensated by high levels of haem O and a CO-reactive cytochrome o in membranes; no other oxidases were detected. In contrast, the wild-type strain had considerable amounts of haem A and negligible levels of haem O. The mutant PYM1 exhibited normal colony morphology, growth, and sporulation in nonfermentable media, whereas on fermentable media, the mutant overproduced acid, which led to poor growth and inhibition of sporulation. External control of the pH of the medium in fermentable media allowed close-to-normal growth and massive sporulation of the mutant. The presence of membrane-bound cytochrome caa 3 -OII and aa 3 -II subunits in strain PYM1 was confirmed by Western blots and haem C staining (COII subunit). Western blotting also revealed that in contrast to the wild-type – strain PYM1 contained the membrane-bound subunits caa 3 -COI and aa 3 -I, but in low amounts. The effect of several respiratory inhibitors on the respiratory system of strain PYM1 suggested that the terminal oxidase is highly resistant to KCN and CO and that a c-type cytochrome might be involved in the electron transfer sequence to the putative cytochrome bo.
Keywords: Key wordsBacillus cereus; Haem O; Cytochrome; bo; Cytochrome aa3; cytochrome caa3; Bacillus; sporulation
Biotransformation of 1-naphthalenesulfonic acid by the green alga Scenedesmus obliquus by H. Kneifel; Kerstin Elmendorff; Eberhard Hegewald; Carl J. Soeder (pp. 32-37).
Under sulfate limitation, axenic batch cultures of the green alga Scenedesmus obliquus metabolized 1-naphthalenesulfonic acid and partially used the sulfonate as a source of sulfur. The main metabolite, 1-hydroxy-2-naphthalenesulfonic acid, which was not metabolized further in the algal culture, was formed by hydroxylation of the substrate in position 1 and by migration of the sulfonic acid group to position 2 of the naphthalene ring (NIH shift). A smaller amount of 1-naphthalenesulfonic acid was desulfonated. The resulting 1-naphthol was mostly transformed into 1-naphthyl β-d-glucopyranoside.
Keywords: Key wordsScenedesmus obliquus; 1-Naphthalenesulfonic acid degradation; NIH shift; Metabolites; 1-Hydroxy-2-naphthalenesulfonic acid; 1-Naphthol; 1-Naphthyl β-d-glucopyranoside
Physiological characteristics and growth behavior of single and double hydrogenase mutants of Desulfovibrio fructosovorans by Souria Malki; Gilles De Luca; Marie Laure Fardeau; M. Rousset; Jean Pierre Belaich; Z. Dermoun (pp. 38-45).
The presence of one periplasmic [NiFe] hydrogenase, one periplasmic [Fe] hydrogenase, and one cytoplasmic NADP-reducing hydrogenase has been previously established in Desulfovibrio fructosovorans. In the present work, marker-exchange mutagenesis was performed to determine the function of the tetrameric NADP-reducing hydrogenase encoded by the hndA, B, C, and D genes. The mutations performed were not lethal to the cells, although the H2-dependent NADP reduction was completely abolished. The double-mutated DM4 (ΔhynABC, ΔhndD) strain was still able to grow on hydrogen plus sulfate as the sole energy source. The growth may have occurred under these culture conditions because of the presence of the remaining [Fe] hydrogenase. The cells grew differently on various substrates depending on whether fructose, lactate, or pyruvate was used in the presence of sulfate. The (hnd mutant growth rates were 25–70% lower than those of the wild-type strain, although the molar growth yield remained unchanged. By contrast, mutants devoid of both [NiFe] hydrogenase and NADP-reducing hydrogenase had 24-38% lower growth yields and showed a corresponding drop in the growth rates. We concluded that each of the three hydrogenases may contribute to the energy supply in D. fructosovorans and that the loss of one enzyme might be compensated for by another. However, the loss of two hydrogenases affected the phosphorylation accompanying the metabolism of fructose, lactate, and pyruvate.
Keywords: Key words Desulfovibrio; Sulfate-reducing bacteria; NADP-reducing hydrogenase; Marker exchange; mutagenesis; Electrotransformation; Growth parameters
Methylosulfonomonas methylovora gen. nov., sp. nov., and Marinosulfonomonas methylotropha gen. nov., sp. nov.: novel methylotrophs able to grow on methanesulfonic acid by Andrew J. Holmes; D. P. Kelly; Simon C. Baker; A. S. Thompson; Paolo De Marco; Elizabeth M. Kenna; J. Colin Murrell (pp. 46-53).
Two novel genera of restricted facultative methylotrophs are described; both Methylosulfonomonas and Marinosulfonomonas are unique in being able to grow on methanesulfonic acid as their sole source of carbon and energy. Five identical strains of Methylosulfonomonas were isolated from diverse soil samples in England and were shown to differ in their morphology, physiology, DNA base composition, molecular genetics, and 16S rDNA sequences from the two marine strains of Marinosulfonomonas, which were isolated from British coastal waters. The marine strains were almost indistinguishable from each other and are considered to be strains of one species. Type species of each genus have been identified and named Methylosulfonomonas methylovora (strain M2) and Marinosulfonomonas methylotropha (strain PSCH4). Phylogenetic analysis using 16S rDNA sequencing places both genera in the α-Proteobacteria. Methylosulfonomonas is a discrete lineage within the α-2 subgroup and is not related closely to any other known bacterial genus. The Marinosulfonomonas strains form a monophyletic cluster in the α-3 subgroup of the Proteobacteria with Roseobacter spp. and some other partially characterized marine bacteria, but they are distinct from these at the genus level. This work shows that the isolation of bacteria with a unique biochemical character, the ability to grow on methanesulfonic acid as energy and carbon substrate, has resulted in the identification of two novel genera of methylotrophs that are unrelated to any other extant methylotroph genera.
Keywords: Key wordsMethylosulfonomonas methylovora; Marinosulfonomonas methylotropha; Methanesulfonic; acid; 16S rRNA phylogeny; 16S rDNA sequences; Proteobacteria
Clostridium vincentii sp. nov., a new obligately anaerobic, saccharolytic, psychrophilic bacterium isolated from low-salinity pond sediment of the McMurdo Ice Shelf, Antarctica by D. O. Mountfort; Frederick A. Rainey; Jutta Burghardt; Heinrich F. Kaspar; Erko Stackebrandt (pp. 54-60).
A gram-positive, motile, rod-shaped, strictly anaerobic bacterium was isolated from an enrichment initiated with sediment taken from below the cyanobacterial mat of a low-salinity pond on the McMurdo Ice Shelf, Antarctica. The organism grew optimally at 12° C, at pH 6.5, and at an NaCl concentration of < 0.5% (w/v). It survived freeze-thawing at low salt concentrations, but not exposure to temperatures over 25° C for more than 20 h or short-term exposure to temperatures > 50° C. Out of a variety of polysaccharides tested as growth substrates, only xylan supported growth. The organism also grew on a variety of mono- and disaccharides including the cyanobacterial cell wall constituent, N-acetyl glucosamine. Fermentation products on a mol product per 100 mol of hexose monomer fermented basis were: acetate, 72; formate, 72; butyrate, 55; hydrogen, 114; and CO2, 100. Not detectable in the culture medium (< 2 mol per 100 mol of monomer) were lactate, propionate, ethanol, n-propanol, n-butanol, and succinate. The G+C content of the DNA from the bacterium was 33 mol%, and a phylogenetic analysis indicated that it grouped closely with members of the RNA-DNA homology group 1 of the genus Clostridium. It differed from other species of this genus with regard to growth temperature optimum, substrate range, and fermentation pattern, and is therefore designated as a new species of Clostridium for which the name Clostridium vincentii is proposed. The type strain is lac-1 (DSM 10228).
Keywords: Key wordsClostridium vincentii; Psychrophile; Obligate anaerobe-saccharophile; Antarctica
Identification of an assimilatory nitrate reductase in mutants of Paracoccus denitrificans GB17 deficient in nitrate respiration by Heather J. Sears; Phillip J. Little; D. J. Richardson; B. C. Berks; Stephen Spiro; Stuart J. Ferguson (pp. 61-66).
A Paracoccus denitrificans strain (M6Ω) unable to use nitrate as a terminal electron acceptor was constructed by insertional inactivation of the periplasmic and membrane-bound nitrate reductases. The mutant strain was able to grow aerobically with nitrate as the sole nitrogen source. It also grew anaerobically with nitrate as sole nitrogen source when nitrous oxide was provided as a respiratory electron acceptor. These growth characteristics are attributed to the presence of a third, assimilatory nitrate reductase. Nitrate reductase activity was detectable in intact cells and soluble fractions using nonphysiological electron donors. The enzyme activity was not detectable when ammonium was included in the growth medium. The results provide an unequivocal demonstration that P. denitrificans can express an assimilatory nitrate reductase in addition to the well-characterised periplasmic and membrane-bound nitrate reductases.
Keywords: Key words Nitrate assimilation; Nitrate reductase; Paracoccus denitrificans; Thiosphaera pantotropha