Check out our New Publishers' Select for Free Articles

Archives of Microbiology (v.169, #3)

New roles for CO2 in the microbial metabolism of aliphatic epoxides and ketones by S. A. Ensign; Frederick J. Small; Jeffrey R. Allen; Miriam K. Sluis (pp. 179-187).

Short-chain aliphatic epoxides and ketones are two classes of toxic organic compounds formed biogenically and anthropogenically. In spite of their toxicity, these compounds are utilized as primary carbon and energy sources or are generated as intermediate metabolites in the metabolism of other compounds (e.g., alkenes, alkanes, and secondary alcohols) by a number of diverse bacteria. One bacterium capable of using both classes of compounds is the gram-negative aerobe Xanthobacter strain Py2. Studies of epoxide and ketone (acetone) metabolism by Xanthobacter strain Py2 have revealed a central role for CO₂ in these processes. Both classes of compounds are metabolized by carboxylation reactions that produce β-keto acids as products. The epoxide- and ketone-converting enzymes are distinct carboxylases with molecular properties and cofactor requirements unprecedented for other carboxylases. Epoxide carboxylase is a four-component multienzyme complex that requires NADPH and NAD⁺ as cofactors. In the course of epoxide carboxylation, a transhydrogenation reaction occurs wherein NADPH undergoes oxidation and NAD⁺ undergoes reduction. Acetone carboxylase is a multimeric (three-subunit) ATP-dependent enzyme that forms AMP and inorganic phosphate as ATP hydrolysis products in the course of acetone carboxylation. Recent studies have demonstrated that acetone metabolism in diverse anaerobic bacteria (sulfate reducers, denitrifiers, phototrophs, and fermenters) also proceeds by carboxylation reactions. ATP-dependent acetone carboxylase activity has been demonstrated in cell-free extracts of the anaerobic acetone-utilizers Rhodobacter capsulatus, Rhodomicrobium vannielii, and Thiosphaera pantotropha. These studies have identified new roles for CO₂ as a cosubstrate in the metabolism of two classes of important xenobiotic compounds. In addition, two new classes of carboxylases have been identified, the investigation of which promises to reveal new insights into biological strategies for the fixation of CO₂ to organic substrates.

Keywords: Key words Acetone; Epoxide; Epoxyalkane; Carboxylase; CO2 fixation; Bacterial metabolism; Xanthobacter

Structural and immunochemical characterization of β-1,2-linked mannobiosyl phosphate residue in the cell wall mannan of Candida glabrata by H. Kobayashi; Hiroko Oyamada; Naoko Iwadate; Hiromi Suzuki; Hideko Mitobe; Kaori Takahashi; Nobuyuki Shibata; Shigeo Suzuki; Yoshio Okawa (pp. 188-194).

A mannan of Candida glabrata IFO 0622 digested by Arthrobacter exo-α-mannosidase and a β-1,2-linked mannobiose obtained from the parent mannan by acid treatment was analyzed using ¹³C nuclear magnetic resonance spectroscopy. The results show that the β-1,2-linked mannobiosyl residue is esterified to a phosphate group through position C-1 in the α-configuration, Manβ1– 2Manα1–HPO₃–. The results of immunochemical assays of these mannans using the commercial antigenic factor sera of the genus Candida (Candida Check, Iatron) indicate that the main recognition site of serum no. 6 in this kit is the mannotetraosyl side-chain Manβ1–2Manα1– 2Manα1–2Man in C. glabrata mannan and also suggest that the phosphate-containing unit (such as Manβ1– 2Manα1–HPO₃– in this mannan) behaves as one of the antigenic determinants of serum no. 6, but not of serum no. 5. Therefore, the present and previous findings indicate that serum no. 5 recognizes relatively longer β-1,2-linked oligomannosyl side-chains, Manβ1–[2Manβ1–]_n 2Man (n = 1–6), attached to the phosphate groups previously observed in the cell wall mannans of Candida albicans, Candida stellatoidea, and Candida tropicalis.

Keywords: Key wordsCandida glabrata; Cell wall mannan; Polysaccharide antigen; Antigenic factor; Chemical; structure; β-1; 2 Linkage; Phosphodiester; 13C nuclear; magnetic resonance

Two internal pools of soluble polyphosphate in the cyanobacterium Synechocystis sp. strain PCC 6308: an in vivo 31P NMR spectroscopic study by B. A. Lawrence; C. Suarez; A. DePina; Eleanor Click; Nancy H. Kolodny; M. M. Allen (pp. 195-200).

Two intracellular pools of soluble polyphosphate were identified by in vivo ³¹P NMR spectroscopy in the cyanobacterium Synechocystis sp. strain PCC 6308. Polyphosphate was present in the cells after growth in sulfur-limited media containing excess phosphate. The presence of polyphosphate was confirmed by transmission electron microscopy and chemical analysis. ³¹P NMR spectroscopy of whole cells treated with EDTA revealed two pools of mobile polyphosphate. A downfield shift and narrowing of part of the broad polyphosphate resonance was observed after EDTA treatment, suggesting that EDTA binds metal ions normally associated with some of the polyphosphate. Phosphate, but not polyphosphate, leaked out of the cells after this treatment. Addition of magnesium ions caused the downfield shift in the polyphosphate resonance to move back toward its original value. These data show that only part of the cation-complexed polyphosphate is accessible to the added EDTA and suggest that there are two internal fractions of NMR-visible polyphosphate in the cells, only one of which loses its associated cations to EDTA. Spheroplast formation showed that polyphosphate was not present in the periplasm of the cells.

Keywords: Key words Cyanobacteria; Polyphosphate; In vivo 31P NMR spectroscopy; Synechocystis sp. strain PCC 6308; EDTA permeabilization; Spheroplasts

Two F420-reducing hydrogenases in Methanosarcina barkeri by Martin Vaupel; R. K. Thauer (pp. 201-205).

F₄₂₀-reducing hydrogenases are nickel iron-sulfur flavoproteins involved in CO₂ reduction with H₂ to methane in methanogenic archaea. Evidence is presented that Methanosarcina barkeri contains two isoenzymes for which the encoding genes have been cloned and sequenced. The genes are organized in two operons, frhADGB and freAEGB, each comprising four open reading frames. Transcription analysis revealed that both operons are transcribed during growth of Ms. barkeri on H₂/CO₂, on methanol, and on trimethylamine, but not during growth on acetate.

Keywords: Key words Methanogenic archaea; Methanosarcina barkeri; Hydrogenases; Coenzyme F420; Gene; expression

Function of H2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum in coenzyme F420 reduction with H2 by C. Afting; A. Hochheimer; R. K. Thauer (pp. 206-210).

In most methanogenic archaea, two hydrogenase systems that can catalyze the reduction of coenzyme F₄₂₀ (F₄₂₀) with H₂ are present: (1) the F₄₂₀-reducing hydrogenase, which is a nickel iron-sulfur flavoprotein composed of three different subunits, and (2) the N ⁵, N¹⁰-methylenetetrahydromethanopterin dehydrogenase system, which is composed of H₂-forming methylenetetrahydromethanopterin dehydrogenase and F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase, both metal-free proteins without an apparent prosthetic group. We report here that in nickel-limited chemostat cultures of Methanobacterium thermoautotrophicum, the specific activity of the F₄₂₀-reducing Ni/Fe-hydrogenase was essentially zero, whereas that of the H₂-forming methylenetetrahydromethanopterin dehydrogenase was six times higher, and that of the F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase was four times higher than in cells grown under non-nickel-limited conditions. This evidence supports the hypothesis that when M. thermoautotrophicum grows under conditions of nickel limitation, the reduction of F₄₂₀ with H₂ is catalyzed by the metal-free methylenetetrahydromethanopterin dehydrogenase system.

Keywords: Key words Hydrogenases; Coenzyme F420; N5; N10-Methylenetetrahydromethanopterin; Methanobacterium thermoautotrophicum; Nickel-limited chemostat culture

Characterization of the alternative σ-factors SigD and SigE in Synechococcus sp. strain PCC 7002. SigE is implicated in transcription of post-exponential-phase-specific genes by T. M. Gruber; D. A. Bryant (pp. 211-219).

The sigD and sigE genes, which encode two alternative σ-factors from the unicellular marine cyanobacterium Synechococcus sp. PCC 7002, were cloned and characterized. Strains in which the sigD and sigE genes were insertionally inactivated were viable under standard laboratory conditions, indicating that SigD and SigE are group 2 σ-factors. When stationary-phase cells were diluted into fresh growth medium, it was observed that the sigE mutant strain required longer times to re-establish exponential growth than the wild-type strain. By monitoring the growth rates in such dilution experiments, it was observed that the lag times for the mutant strain became progressively longer as the original cultures progressed towards stationary phase. Transcripts for the sigE gene initially increased and subsequently decreased as cells grew further into stationary phase. It was determined that a functional SigE protein is required for the expression of the starvation-induced protein DpsA/PexB. The results suggest that SigE is involved in the transcription of genes specifically expressed in the post-exponential phase.

Keywords: Key wordsSynechococcus sp. PCC 7002; Cyanobacterium; σ-Factor; RNA polymerase; RpoS; DpsA; PexB; Stationary phase

Thermostable phenylalanine dehydrogenase from a mesophilic Microbacterium sp. strain DM 86-1 by Y. Asano; Masako Tanetani (pp. 220-224).

Bacteria that produced NAD⁺-dependent phenylalanine dehydrogenase (EC 1.4.1.20) were selected among l-methionine utilizers isolated from soil. A bacterial strain showing phenylalanine dehydrogenase activity was chosen and classified in the genus Microbacterium. Phenylalanine dehydrogenase was purified from the crude extract of Microbacterium sp. strain DM 86-1 (TPU 3592) to homogeneity as judged by SDS-polyacrylamide disc gel electrophoresis. The enzyme has an isoelectric point of 5.8 and a relative molecular weight (M _r) of approximately 330,000. The enzyme is composed of eight identical subunits with an M _r of approximately 41,000. The apparent K _m values for l-phenylalanine and NAD⁺ were calculated to be 0.10 mM and 0.20 mM, respectively. No loss of the enzyme activity was observed upon incubation at 55° C for 10 min.

Keywords: Key words Phenylalanine dehydrogenase; Microbacterium sp. strain DM 86-1

Isolation and immunocytochemical location of the nitrite-oxidizing system in Nitrospira moscoviensis by E. Spieck; Silke Ehrich; Jens Aamand; Eberhard Bock (pp. 225-230).

A membrane-associated nitrite-oxidizing system of Nitrospira moscoviensis was isolated from heat-treated membranes. The four major proteins of the enzyme fraction had apparent molecular masses of 130, 62, 46, and 29 kDa, respectively. The nitrite-oxidizing activity was dependent on the presence of molybdenum. In contrast to the nitrite oxidoreductase of Nitrobacter hamburgensis X14, the activity of the nitrite-oxidizing system of Ns. moscoviensis increased when solubilized by heat treatment. Electron microscopy of the purified enzyme revealed uniform particles with a size of approximately 7 × 9 nm. SDS-immunoblotting analysis of crude extracts showed that the monoclonal antibodies Hyb 153–3, which recognize the β-subunit of the nitrite oxidoreductase from Nitrobacter, reacted with a protein of 50 kDa in Ns. moscoviensis. This protein corresponded to the protein of 46 kDa of the purified enzyme and contained a b-type cytochrome. Using electron microscopic immunocytochemistry and the monoclonal antibodies Hyb 153–3, the nitrite-oxidizing system of Ns. moscoviensis was shown to be located in the periplasmic space. Here a periodic arrangement of membrane-associated particles was found on the outside of the cytoplasmic membrane in the form of a hexagonal pattern. It is supposed that these particles represent the nitrite-oxidizing system in Nitrospira.

Keywords: Key wordsNitrospira moscoviensis; Nitrite-oxidizing system; Membrane-associated enzyme; Periplasmic space; Monoclonal antibodies; Post-embedding labeling; Hexagonal pattern

Psychromonas antarcticus gen. nov., sp. nov., a new aerotolerant anaerobic, halophilic psychrophile isolated from pond sediment of the McMurdo Ice Shelf, Antarctica by D. O. Mountfort; Frederick A. Rainey; Jutta Burghardt; Heinrich F. Kaspar; Erko Stackebrandt (pp. 231-238).

A gram-negative, rod- to oval-shaped, aerotolerant anaerobic bacterium was isolated from an anaerobic enrichment inoculated with sediment taken from below the cyanobacterial mat of a high-salinity pond near Bratina Island on the McMurdo Ice Shelf, Antarctica. The organism was positive for terminal oxidase and catalase and was motile by means of a polar flagellum. Optimal growth of anaerobic cultures occurred at 12° C, at pH 6.5, and at an NaCl concentration of 3% (w/v). Of a variety of polysaccharides tested, only starch and glycogen supported growth. No growth was observed on cellulosic substrates and xylan, and the organism was unable to attack esculin. Monosaccharides and disaccharides, including the cyanobacterial cell-wall constituent N-acetyl glucosamine, were fermented. Per 100 mol of hexose, the following products (in mol) were formed: acetate, 60; formate, 130; ethanol, 56; lactate, 73; CO₂, 15; and butyrate, 2. Propionate, ethanol, n-propanol, n-butanol and succinate were not detectable in the culture medium (< 1 mol per 100 mol of monomer). Hydrogen was not detected in the head space (detection limit < 10^–5 atm). Growth yields in aerobic static liquid cultures were slightly higher than those in anaerobic culture, and fermentation favoured acetate at the expense of electron sink products. Growth was inhibited in aerobic shaking cultures, and the organism did not utilize nitrate or sulfate as electron acceptors. The G+C content of the DNA from the bacterium was 42.8 mol%. A phylogenetic analysis indicated that the organism is a member of the γ-subgroup of Proteobacteria, but that it is distinct from other members of this group based on the sequence of its 16S rRNA gene, mol% G+C, morphology, and physiological and biochemical characteristics. It is designated as a new genus and species; the type strain is star-1 (DSM 10704).

Keywords: Key wordsPsychromonas antarcticus; Psychrophile; Aerotolerant anaerobe; Saccharophile; Antarctica

Reduction of diverse electron acceptors by Aeromonas hydrophila by V. Knight; R. Blakemore (pp. 239-248).

Aeromonas hydrophila ATCC 7966 grew anaerobically on glycerol with nitrate, fumarate, Fe(III), Co(III), or Se(VI) as the sole terminal electron acceptor, but did not ferment glycerol. Final cell yields were directly proportional to the amount of terminal electron acceptor provided. Twenty-four estuarine mesophilic aeromonads were isolated; all reduced nitrate, Fe(III), or Co(III), and five strains reduced Se(VI). Dissimilatory Fe(III) reduction by A. hydrophila may involve cytochromes. Difference spectra obtained with whole cells showed absorption maxima at wavelengths characteristic of c-type cytochromes (419, 522, and 553 nm). Hydrogen-reduced cytochromes within intact cells were oxidized by the addition of Fe(III) or nitrate. Studies with respiratory inhibitors yielded results consistent with a respiratory chain involving succinate (flavin-containing) dehydrogenase, quinones and cytochromes, and a single Fe(III) reductase. Neither anaerobic respiration nor dissimilatory metal reduction by members of the genus Aeromonas have been reported previously.

Keywords: Key words Nitrate reduction; Fe(III) reduction; Co(III) reduction; Se(VI) reduction; Mesophilic aeromonads; Cytochromes; Estuarine bacteria

Chemolithoautotrophy and mixotrophy in the thiophene-2-carboxylic acid-utilizing Xanthobacter tagetidis by A. Nikki Padden; Donovan P. Kelly; A. P. Wood (pp. 249-256).

Xanthobacter tagetidis grew as a chemolithotrophic autotroph on thiosulfate and other inorganic sulfur compounds, as a heterotroph on thiophene-2-carboxylic acid, acetic acid and α-ketoglutaric acid, and as a mixotroph on thiosulfate in combination with thiophene-2-carboxylic acid and/or acetic acid. Autotrophic growth on one-carbon organosulfur compounds, and intermediates in their oxidation are also reported. Thiosulfate enhanced the growth yields in mixotrophic cultures, presumably by acting as a supplementary energy source, since ribulose bisphosphate carboxylase was only active in thiosulfate-grown cells and was not detected in mixotrophic cultures using thiosulfate with thiophene-2-carboxylic acid. Bacteria grown on thiophene-2-carboxylic acid also oxidized sulfide, thiosulfate and tetrathionate, indicating these as possible sulfur intermediates in thiophene-2-carboxylic acid degradation. Thiosulfate and tetrathionate were oxidized completely to sulfate and, consequently, did not accumulate as products of thiophene-2-carboxylic acid oxidation in growing cultures. K _m and V _max values for the oxidation of thiosulfate, tetrathionate or sulfide were 13 μM and 83 nmol O₂ min^–1 (mg dry wt.)^–1, respectively; thiosulfate and tetrathionate became autoinhibitory at concentrations above 100 μM. The true growth yield (Y_max) on thiophene-2-carboxylic acid was estimated from chemostat cultures (at dilution rates of 0.034–0.094 h^–1) to be 112.2 g mol^–1, with a maintenance coefficient (m) of 0.3 mmol thiophene-2-carboxylic acid (g dry wt.)^–1 h^–1, and the maximum specific growth rate (μ_max) was 0.116 h^–1. Growth in chemostat culture at a dilution rate of 0.041 h^–1 indicated growth yields [g dry wt. (mol substrate)^–1] of 8.1 g (mol thiosulfate)^–1, 60.9 g (mol thiophene-2-carboxylic acid)^–1, and 17.5 g (mol acetic acid)^–1, with additive yields for growth on mixtures of these substrates. At a dilution rate of 0.034 h^–1, yields of 57.8 g (mol α-ketoglutaric acid)^–1 and 60.7 g (mol thiophene-2-carboxylic acid)^–1 indicated some additional energy conservation from oxidation of the thiophene-sulfur. SDS-PAGE of cell-free preparations indicated a polypeptide (M _r, 21.0 kDa) specific to growth on thiophene-2-carboxylic acid for which no function can yet be ascribed: no metabolism of thiophene-2-carboxylic acid by cell-free extracts was detected. It was shown that X. tagetidis exhibits a remarkable degree of metabolic versatility and is representative of facultatively methylotrophic and chemolithotrophic autotrophs that contribute significantly to the turnover of simple inorganic and organic sulfur compounds (including substituted thiophenes) in the natural environment.

Keywords: Key wordsXanthobacter tagetidis; Thiophene-2-carboxylic acid oxidation; Autotrophy; Mixotrophy; Sulfur compound oxidation; Chemostat culture kinetics

Enzyme analyses demonstrate that β-methylbutyric acid is converted to β-hydroxy-β-methylbutyric acid via the leucine catabolic pathway by Galactomyces reessii by In-Young Lee; J. P. N. Rosazza (pp. 257-262).

Galactomyces reessii accomplishes the enzymatic transformation of β-methylbutyric acid (isovaleric acid) to β-hydroxy-β-methylbutyric acid. The enzymatic basis for this bioconversion was evaluated by analyzing cell-free extracts of G. reessii for enzyme activities commonly associated with leucine catabolism. G. reessii extracts contained activities for acyl-CoA synthetase, acyl-CoA dehydrogenase, and enoyl-CoA hydratase, whereas β-methylbutyric acid hydroxylase, α-ketoisocaproate oxygenase, and acyl-CoA oxidase (with isovaleryl-CoA as substrate) were not observed. Furthermore, β-methylbutyric acid is initially activated to isovaleryl-CoA by acyl-CoA synthetase, dehydrogenated to methylcrotonyl-CoA by acyl-CoA dehydrogenase, hydrated to β-hydroxy-β-methylbutyric acid-CoA by enoyl-CoA hydratase, and hydrolyzed to β-hydroxy-β-methylbutyric acid in G. reessii extracts. Cell-free extracts converted both isovaleryl-CoA and methylcrotonyl-CoA into β-hydroxy-β-methylbutyric acid, thus demonstrating that β-methylbutyric acid is part of the leucine catabolic pathway. The rate of β-methylbutyric acid conversion to β-hydroxy-β-methylbutyric acid with cell-free extract was 0.013 μmol β-hydroxy-β-methylbutyric acid (mg protein)^–1 h^–1, while the conversion rate of leucine was fivefold lower. With whole cells, the highest production rate [0.042 μmol β-hydroxy-β-methylbutyric acid (g cells)^–1 h^–1] was also observed with β-methylbutyric acid. The results indicate that β-methylbutyric acid is transformed to β-hydroxy-β-methylbutyric acid through the leucine catabolic pathway.

Keywords: Key wordsβ-hydroxy-β-methylbutyric acid; Metabolism; Galactomyces reessii

Alkali-labile precursors of dimethyl sulfide in marine benthic cyanobacteria by C. Vogt; Andreas Rabenstein; Jörg Rethmeier; Ulrich Fischer (pp. 263-266).

By the method of cold alkali hydrolysis, 29 marine benthic cyanobacteria were screened for production of alkali-labile precursors of dimethyl sulfide (DMS) including dimethylsulfoniopropionate (DMSP), a compound of significant importance in marine environments. Concentrations of DMS precursors ranged from undetectable to 0.8 mmol (g Chl a)^–1. The data correspond to some previous investigations concerning DMSP content of marine cyanobacteria and suggest that marine benthic cyanobacteria are only minor producers of DMSP.

Keywords: Key words Marine cyanobacteria; Dimethylsulfoniopropionate; Dimethyl sulfide

Sections

Personal tools

Archives of Microbiology (v.169, #3)

Sections

Personal tools

Local resources

Archives of Microbiology (v.169, #3)