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Archives of Microbiology (v.168, #5)

Molybdate transport and regulation in bacteria by A. M. Grunden; K. T. Shanmugam (pp. 345-354).

Molybdate is transported in bacteria by a high-affinity transport system composed of a periplasmic binding protein, an integral membrane protein, and an energizer protein. These three proteins are coded by modA, modB, and modC genes, respectively. The ModA, ModB, and ModC proteins from various organisms (Escherichia coli, Haemophilus influenzae, Azotobacter vinelandii, and Rhodobacter capsulatus) are very similar. The lowest K _m value reported for molybdate in the molybdate transport process is approximately 50 nM. In a mod mutant, molybdate is transported by the sulfate transport system or by a nonspecific anion transporter. Molybdate transport is tightly coupled to utilization in E. coli and Klebsiella pneumoniae, while other dinitrogen-fixing organisms appear to have a molybdenum storage protein. In all organisms studied so far, molybdate transport genes are regulated by a repressor protein, ModE. The ModE-molybdate complex binds to the sequences TAYAT (Y = T or C) in the operator/ promoter region in E. coli and prevents transcription of the modABCD operon. The ModE-molybdate complex binds to DNA as a homodimer in E. coli and possibly in other organisms as well. In R. capsulatus, however, two ModE homologues (MopAB proteins) are required for repression.

Keywords: Key words Molybdate transport; modABC Genes; modE Gene; Molybdate-specific repressor; ABC; transport system; Molybdate transport/genetics/; regulation; Escherichia coli; Azotobacter vinelandii; Rhodobacter capsulatus; Clostridium pasteurianum

Isolation of new 6-methylnicotinic-acid-degrading bacteria, one of which catalyses the regioselective hydroxylation of nicotinic acid at position C2 by Andreas Tinschert; Andreas Kiener; Klaus Heinzmann; A. Tschech (pp. 355-361).

2-Hydroxynicotinic acid is an important building block for herbicides and pharmaceuticals. Enrichment strategies to increase the chances of finding microorganisms capable of hydroxylating at the C2 position and to avoid the degradation of nicotinic acid via the usual intermediate, 6-hydroxynicotinic acid, were used. Three bacterial strains (Mena 23/3–3c, Mena 25/4–1, and Mena 25/ 4–3) were isolated from enrichment cultures with 6-methylnicotinic acid as the sole source of carbon and energy. Partial characterization of these strains indicated that they represent new bacterial species. All three strains completely degraded 6-methylnicotinic acid, and evidence is presented that the first step in the degradation pathway of strain Mena 23/3–3c is hydroxylation at the C2 position. Resting cells of this strain grown on 6-methylnicotinic acid also hydroxylated nicotinic acid at the C2 position, but did not further degrade the product. Strain Mena 23/ 3–3c showed the highest degree of 16S rRNA sequence similarity to members of the genera Ralstonia and Burkholderia.

Keywords: Key words 6-Methylnicotinic acid; 2-Hydroxy-6-methylnicotinic acid; Nicotinic acid; 2-Hydroxynicotinic acid; Ralstonia; Burkholderia; Paenibacillus; Agrobacterium; Rhizobium

Cytochrome c peroxidase from Methylococcus capsulatus Bath by James A. Zahn; David M. Arciero; A. B. Hooper; Joel R. Coats; A. A. DiSpirito (pp. 362-372).

A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (E _m7 = –254 mV) and one low-spin, high-potential (E _m7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c ₅₅₅ as the electron donor, the K _M and V _max for peroxide reduction were 510 ± 100 nM and 425 ± 22 mol ferrocytochrome c ₅₅₅ oxidized min^–1 (mole cytochrome c peroxidase)^–1, respectively.

Keywords: Key words Methanotroph; Methylotroph; Cytochrome c peroxidase; Cytochrome aa3; Methylamine oxidation; Methanol oxidation; moxG

Cultivation of hyperthermophilic archaea in capillary tubes resulting in improved preservation of fine structures by Gertraud Rieger; Karin Müller; René Hermann; Karl Otto Stetter; R. Rachel (pp. 373-379).

A method for cultivating hyperthermophilic archaea that results in very high cell densities and in improved structural preservation of the cells is described. Cellulose capillary tubes, originally introduced as containers for embedding for electron microscopy, were filled with cells, closed at both ends, and put into sterile culture medium. Within these capillaries, which serve as ultrafiltration chambers, cells could be cultivated to much higher cell densities than in regular cultures. The capillaries containing cells were processed for ultrathin-sectioning by fixation, freeze-substitution, and embedding. Using this cultivation procedure, centrifugation, which may destroy sensitive structural components, could be avoided, and the cells of hyperthermophilic archaea were well-preserved. These undisturbed cells revealed the following new structural features: (1) a high number of tubules in ultrathin-sections, indicating a well-preserved network of Pyrodictium cells and tubules; (2) “ultraflat areas” of Pyrodictium cells, with the two membranes being in direct contact and, at some places, bulging out, forming evaginations; (3) novel cell-to-cell connections between Thermoproteus cells and, similarly, between Pyrobaculum cells; and (4) a surface coat on Pyrobaculum aerophilum cells. The cultivation procedure offers distinct advantages over conventional techniques and might be applicable for improved electron microscopy of other sensitive microorganisms.

Keywords: Key words Hyperthermophilic archaea; Cultivation; Cellulose capillary tubes; Cryo-fixation; Freeze-substitution; Cell-to-cell connection; Pyrodictium; Thermoproteus; Pyrobaculum

Dissimilatory arsenate and sulfate reduction in Desulfotomaculum auripigmentum sp. nov. by Dianne K. Newman; Erin K. Kennedy; John D. Coates; Dianne Ahmann; Debra J. Ellis; Derek R. Lovley; François M. M. Morel (pp. 380-388).

A newly discovered arsenate-reducing bacterium, strain OREX-4, differed significantly from strains MIT-13 and SES-3, the previously described arsenate-reducing isolates, which grew on nitrate but not on sulfate. In contrast, strain OREX-4 did not respire nitrate but grew on lactate, with either arsenate or sulfate serving as the electron acceptor, and even preferred arsenate. Both arsenate and sulfate reduction were inhibited by molybdate. Strain OREX-4, a gram-positive bacterium with a hexagonal S-layer on its cell wall, metabolized compounds commonly used by sulfate reducers. Scorodite (FeAsO₄2· H₂O) an arsenate-containing mineral, provided micromolar concentrations of arsenate that supported cell growth. Physiologically and phylogenetically, strain OREX-4 was far-removed from strains MIT-13 and SES-3: strain OREX-4 grew on different electron donors and electron acceptors, and fell within the gram-positive group of the Bacteria, whereas MIT-13 and SES-3 fell together in the ɛ-subdivision of the Proteobacteria. Together, these results suggest that organisms spread among diverse bacterial phyla can use arsenate as a terminal electron acceptor, and that dissimilatory arsenate reduction might occur in the sulfidogenic zone at arsenate concentrations of environmental interest. 16S rRNA sequence analysis indicated that strain OREX-4 is a new species of the genus Desulfotomaculum, and accordingly, the name Desulfotomaculum auripigmentum is proposed.

Keywords: Key wordsDesulfotomaculum; Arsenate reduction; Sulfate reduction

Immunochemically distinct NADP-linked isocitrate dehydrogenase isozymes in mitochondria and peroxisomes of Candida tropicalis by Toshiya Imajo; Hiroyuki Kawachi; Haruyuki Atomi; Shin-ichi Sanuki; Setsuko Yamamoto; Mitsuyoshi Ueda; A. Tanaka (pp. 389-395).

Although peroxisomal localization of NADP-linked isocitrate dehydrogenase (Idp) was first demonstrated in Candida tropicalis, the mitochondrial isozyme has not been found in this yeast. Here we report that the presence of mitochondrial Idp in the yeast was demonstrated by screening for its gene with a DNA probe containing conserved sequences of Idps from various organisms. The nucleotide sequence of the gene (CtIDP1) revealed a 1,290-bp open reading frame corresponding to a 430-amino-acid protein with a high similarity to previously reported Idps. Overexpression of CtIDP1 in Saccharomyces cerevisiae gave a high intracellular Idp activity, and the purified recombinant Idp was shown to be a homodimer with a subunit molecular mass of approximately 44 kDa, different from that of peroxisomal Idp (45 kDa) previously purified from C. tropicalis. Western blot analysis of the subcellular fractions from acetate-grown C. tropicalis with polyclonal antibodies raised against the recombinant CtIdp1 showed that the CtIdp1 in C. tropicalis was localized in mitochondria but not in peroxisomes. Similar levels of CtIDP1 mRNA and its protein product were detected in cells grown on glucose, acetate, and n-alkane, although a slight decrease was observed in n-alkane-grown cells. From these results, CtIdp1 was demonstrated to be mitochondrial Idp. The properties of mitochondrial Idp and peroxisomal Idp isozymes were proven to be similar, but they were immunochemically distinct, suggesting the presence of another gene responsible for peroxisomal Idp in C. tropicalis.

Keywords: Key wordsCandida tropicalis; NADP-linked isocitrate dehydrogenase; Mitochondria; Peroxisomes; Isozyme

Function of coenzyme F420-dependent NADP reductase in methanogenic archaea containing an NADP-dependent alcohol dehydrogenase by Holger Berk; R. K. Thauer (pp. 396-402).

Methanogenic archaea growing on ethanol or isopropanol as the electron donor for CO₂ reduction to CH₄ contain either an NADP-dependent or a coenzyme F₄₂₀-dependent alcohol dehydrogenase. We report here that in both groups of methanogens, the N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase and the N ⁵, N ¹⁰-methylenetetrahydromethanopterin reductase, two enzymes involved in CO₂ reduction to CH₄, are specific for F₄₂₀. This raised the question how F₄₂₀H₂ is regenerated in the methanogens with an NADP-dependent alcohol dehydrogenase. We found that these organisms contain catabolic activities of an enzyme catalyzing the reduction of F₄₂₀ with NADPH. The F₄₂₀-dependent NADP reductase from Methanogenium organophilum was purified and characterized. The N-terminal amino acid sequence showed 42% sequence identity to a putative gene product in Methanococcus jannaschii, the total genome of which has recently been sequenced.

Keywords: Key words Coenzyme F420; F420-dependent NADP; reductase; F420-dependent N5; N10-methylenetetrahydromethanopterin dehydrogenase; F420-dependent N5; N10-methylenetetrahydromethanopterin reductase; Methanogenic archaea; Methanogenium organophilum; Methanobacterium palustre; Methanogenium liminatans; Methanoculleus; thermophilicus

Characterisation of Escherichia coli K-12 mutants defective in formate-dependent nitrite reduction: essential roles for hemN and the menFDBCE operon by K. Tyson; R. Metheringham; L. Griffiths; J. Cole (pp. 403-411).

Three Escherichia coli mutants defective in formate-dependent nitrite reduction (Nrf activity) were characterised. Two of the mutants, JCB354 and JCB356, synthesized all five c-type cytochromes previously characterised in anaerobic cultures of E. coli. The third mutant, JCB355, was defective for both cytochrome b and cytochrome c synthesis, but only during anaerobic growth. The insertion sites of the transposon in strains JCB354 and JCB356 mapped to the menFDBCE operon; the hemN gene was disrupted in strain JCB355. The mutation in strain JCB354 was complemented by a plasmid encoding only menD; strain JCB356 was complemented by a plasmid encoding only menBCE. A mutant defective in the methyltransferase activity involved in both ubiquinone synthesis and conversion of demethylmenaquinone to menaquinone expressed the same Nrf activity as the parental strain. The effects of men, ubiA and ubiE mutations on other cytochrome-c-dependent electron transfer pathways were also determined. The combined data establish that menaquinones are essential for cytochrome-c-dependent trimethylamine-N-oxide reductase (Tor) and Nrf activity, but that either menaquinone or ubiquinone, but not demethylmenaquinone, can transfer electrons to a third cytochrome-c-dependent electron transfer chain, the periplasmic nitrate reductase.

Keywords: Key words Nitrite reductase mutants; Formate-dependent nitrite reduction; Anaerobic electron transfer; Menaquinones; hemN; Cytochrome c biosynthesis

Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus by Ida Helene Steen; Torleiv Lien; Nils-Kåre Birkeland (pp. 412-420).

A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE. Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity with a high preference for NADP⁺. Optimal temperature for activity was 90° C or above, and a half-life of 22 min was found for the enzyme when incubated at 90° C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into three distinct phylogenetic groups.

Keywords: Key words Isocitrate dehydrogenase; icd; Archaea; Archaeoglobus fulgidus; Thermostable protein; Thermostability; Thermophiles; Hyperthermophiles; Phylogeny

The path of unspecific incorporation of selenium in Escherichia coli by Sabine Müller; Johann Heider; A. Böck (pp. 421-427).

The path of unspecific selenium incorporation into proteins was studied in Escherichia coli mutants blocked in the biosynthesis of cysteine and methionine or altered in its regulation. Selenium incorporation required all enzymatic steps of cysteine biosynthesis except sulfite reduction, indicating that intracellular reduction of selenite occurs nonenzymatically. Cysteine (but not methionine) supplementation prevented unspecific incorporation of selenium by repressing cysteine biosynthesis. On the other hand, when the biosynthesis of cysteine was derepressed in regulatory mutants, selenium was incorporated to high levels. These findings and the fact that methionine auxotrophic strains still displayed unspecific incorporation show that selenium incorporation into proteins in E. coli occurs mainly as selenocysteine. These findings also provide information on the labeling conditions for incorporating ⁷⁵Se only and specifically into selenoproteins.

Keywords: Key wordsEscherichia coli; Selenoproteins; Selenocysteine; Selenomethionine; Monoselenophosphate synthetase; Cysteine biosynthesis

Propionate oxidation in Escherichia coli: evidence for operation of a methylcitrate cycle in bacteria by Susanne Textor; Volker F. Wendisch; Albert A. De Graaf; U. Müller; Monica I. Linder; Dietmar Linder; W. Buckel (pp. 428-436).

Escherichia coli grew in a minimal medium on propionate as the sole carbon and energy source. Initially a lag phase of 4–7 days was observed. Cells adapted to propionate still required 1–2 days before growth commenced. Incorporation of (2-¹³C), (3-¹³C) or (²H₃)propionate into alanine revealed by NMR that propionate was oxidized to pyruvate without randomisation of the carbon skeleton and excluded pathways in which the methyl group was transiently converted to a methylene group. Extracts of propionate-grown cells contained a specific enzyme that catalyses the condensation of propionyl-CoA with oxaloacetate, most probably to methylcitrate. The enzyme was purified and identified as the already-known citrate synthase II. By 2-D gel electrophoresis, the formation of a second propionate-specific enzyme with sequence similarities to isocitrate lyases was detected. The genes of both enzymes were located in a putative operon with high identities (at least 76% on the protein level) with the very recently discovered prp operon from Salmonella typhimurium. The results indicate that E. coli oxidises propionate to pyruvate via the methylcitrate cycle known from yeast. The ¹³C patterns of aspartate and glutamate are consistent with the further oxidation of pyruvate to acetyl-CoA. Oxaloacetate is predominantly generated via the glyoxylate cycle rather than by carboxylation of phosphoenolpyruvate.

Keywords: Key words Escherichia coli; Propionate oxidation; 13C and 2H-labeling; Methylcitrate cycle; Glyoxylate; cycle

Pertactin antigens extracted from Bordetella pertussis and Bordetella bronchiseptica differ in the isoelectric point by Cristina Pagliaccia; Roberto Manetti; R. Rappuoli (pp. 437-440).

Pertactin, which is a membrane-associated antigen of Bordetella pertussis and which is present in many acellular vaccines against whooping cough, has been reported to be similar to the homologous protein in Bordetella bronchiseptica. By running parallel experiments using proteins derived from the two species, we show that the isoelectric point of pertactin from B. pertussis is lower than reported and clearly distinguishable from the homologous protein of B. bronchiseptica.

Keywords: Key words Pertactin; 69K antigen; Bordetella; pertussis; Bordetella bronchiseptica; Pertussis vaccine; Acellular pertussis vaccine

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Archives of Microbiology (v.168, #5)