Showing papers in "Archives of Microbiology in 1989"
TL;DR: A new, rod-shaped, Gram-negative, non-sporing sulfate reducer was enriched and isolated from marine sediment with aniline as sole electron donor and carbon source and degraded anilines completely to CO2 and NH3 with stoichiometric reduction of sulfate to sulfide.
Abstract: A new, rod-shaped, Gram-negative, non-sporing sulfate reducer (strain Ani1) was enriched and isolated from marine sediment with aniline as sole electron donor and carbon source. The strain degraded aniline completely to CO2 and NH3 with stoichiometric reduction of sulfate to sulfide. Strain Ani1 also degraded aminobenzoates and further aromatic and aliphatic compounds. The strain grew in sulfide-reduced mineral medium supplemented only with vitamin B12 and thiamine. Cells contained cytochromes, carbon monoxide dehydrogenase, and sulfite reductase P 582, but no desulfoviridin. Strain Ani1 is described as a new species of the genus Desulfobacterium, D. anilini. Marine enrichments with the three dihydroxybenzene isomers led to three different strains of sulfate-reducing bacteria; each of them could grow only with the isomer used for enrichment. Two strains isolated with catechol (strain Cat2) or resorcinol (strain Re10) were studied in detail. Both strains oxidized their substrates completely to CO2 and contained cytochromes, carbon monoxide dehydrogenase, and sulfite reductase P 582. Desulfoviridin was not present. Whereas the rod-shaped catechol oxidizer (strain Cat2) was able to grow on 18 aromatic compounds and several aliphatic substrates, the coccoid resorcinol-degrading bacterium (strain Re10) utilized only resorcinol, 2,4-dihydroxybenzoate and 1,3-cyclohexanedion. These strains could not be affiliated with existing species of sulfate-reducing bacteria. A further coccoid sulfate-reducing bacterium (strain Hy5) was isolated with hydroquinone and identified as a subspecies of Desulfococcus multivorans. Most-probable-number enumerations with catechol, phenol, and resorcinol showed relatively large numbers (104–106 per ml) or aryl compound-degrading sulfate reducers in marine sediment samples.
172 citations
TL;DR: Curcumin binding to cells was not required for photokilling; the reactive intermediate therefore must be relatively long-lived, and the mechanism(s) of curcumin phototoxicity may involve hydrogen peroxide production.
Abstract: Curcumin is a yellow-orange compound derived from the root of Curcuma longa (Zingiberaceae family), that has been used as a medicine, spice and coloring agent. Curcumin has proved nontoxic in a number of cell culture and whole animal studies. Curcumin has, however, been reported to have bactericidal effects at very high concentrations. When illuminated, curcumin exerted potent phototoxic effects in micromolar amounts. Gram-negative bacteria displayed greater resistance to curcumin phototoxicity relative to Gram-positive bacteria. Oxygen was required for curcumin phototoxicity. Curcumin binding to cells was not required for photokilling; the reactive intermediate therefore must be relatively long-lived. The mechanism(s) of curcumin phototoxicity may involve hydrogen peroxide production. Singlet excited oxygen was not detected.
145 citations
TL;DR: It is suggested that 3HP is an intermediate in a pathway for acetate assimilation and in a new reductive carboxylic acid cycle for autotrophic CO2 fixation.
Abstract: Chloroflexus aurantiacus OK-70 fl secreted 3-hydroxypropionate (3HP) during phototrophic growth. The greatest amounts were secreted by cells grown on propionate (0.35 mM 3HP) while the lowest levels were found in autotrophically grown cultures (1.5 μM). Large amounts of 2-fluoro,3-hydroxypropionate were formed by autotrophically grown cells exposed to fluoroacetate (FAc). Increased levels of 3HP were observed in these cultures when incubated with acctate. The secretion of 3HP was further stimulated by 0.2 mM KCN, an inhibitor of CO2 fixation, but only in the presence of acetate. The pathway of 3HP formation was studied by using 13C-labelled substrates and NMR. The 3HP formed in the presence of C1-labelled acetate and FAc was labelled at C3 and somewhat less at C2 while with C2-labelled acetate as the tracer 3HP was labelled predominantly at C2. The carboxyl group was derived from CO2. The 3HP formed by cells grown on propionate and 13CO2 was labelled at all carbon atoms, the label content of C2 and C3 was about 25 and 65% of that of C1 respectively. It is suggested that 3HP is an intermediate in a pathway for acetate assimilation and in a new reductive carboxylic acid cycle for autotrophic CO2 fixation.
141 citations
TL;DR: The present theory is that maturation of Pep5 involves (a) enzymic conversion of Thr, Ser and Cys into dehydrated amino acids and sulfide bridges, (b) membrane translocation and cleavage of the modified prepeptide, which proves that Pep5 is ribosomally synthesized.
Abstract: A wobbled 14-mer oligonucleotide was derived from the amino acid sequence of the 34-residue propeptide of the lantibiotic Pep5 (Kellner et al. 1989). Using this hybridization probe, the structural gene of Pep5, pepA, was located on the 18.6 kbp plasmid pED503. The nucleotide sequence of pepA codes for a prepeptide with 60 residues and proves that Pep5 is ribosomally synthesized. The N-terminus of the prepeptide has a high α-helix probability and a characteristic proteolytic cleavage site precedes the C-terminal 34-residue propeptide. Our present theory is that maturation of Pep5 involves (a) enzymic conversion of Thr, Ser and Cys into dehydrated amino acids and sulfide bridges, (b) membrane translocation and cleavage of the modified prepeptide.
137 citations
TL;DR: The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later.
Abstract: Two biosynthetic pathways are known for the universal tetrapyrrole precursor, δ-aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO2. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNAGlu, ATP, Mg2+, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[3H]glutamate or 1-[14C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[14C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the α subgroup of purple bacteria.
135 citations
TL;DR: Three new strains of eubacterial hyperthermophiles were isolated from continental solfataric springs at Lac Abbé (Djibouti, Africa) and one of them is described as the new species Thermotoga thermarum.
Abstract: Three new strains of eubacterial hyperthermophiles were isolated from continental solfataric springs at Lac Abbe (Djibouti, Africa). Due to their morphology, lipids, and RNA polymerases they belong to the genus Thermotoga. Strains LA4 and LA10 are closely related to Thermotoga neapolitana found up to now only in the marine environment. Strain LA 3 differs from Thermotoga maritima and Thermotoga neapolitana in significant physiological and molecular properties. It is described as the new species Thermotoga thermarum.
131 citations
TL;DR: Analysis of mutants of Anabaena sp.
Abstract: Mutants of Anabaena sp. PCC 7120 with O2-sensitive acetylene-reducing activity were studied to identify envelope components that contribute to the barrier limiting diffusion of oxygen into the heterocyst. Mutant strain EF114, deficient in a heterocyst-specific glycolipid, reduced acetylene only under strictly anaerobic conditions. Analysis of in vivo O2 uptake as a function of dissolved pO2 showed that EF114 has lost the low affinity, diffusion-limited respiratory component associated with heterocysts in wild-type filaments. The low affinity respiratory activity was also lost in EF116, a mutant in which the cohesiveness of the outer polysaccharide layer was reduced. Restoration of aerobic nitrogen fixation in a spontaneous revertant of EF116 and in a strain complemented with cosmid 41E11 was associated with restoration of the low affinity component of respiratory activity. The results provide evidence that the barrier to diffusion of gas into heterocysts depends upon both the glycolipid layer and the polysaccharide layer of the heterocyst envelope.
128 citations
TL;DR: It is proposed that during thiosulfate and sulfite disproportionation sulfate is formed via APS reductase and ATP sulfurylase, but not by sulfite oxidoreductase.
Abstract: Disproportionation of thiosulfate or sulfite to sulfate plus sulfide was found in several sulfate-reducing bacteria. Out of nineteen strains tested, eight disproportionated thiosulfate, and four sulfite. Growth with thiosulfate or sulfite as the sole energy source was obtained with three strains (Desulfovibrio sulfodismutans and the strains Bra02 and NTA3); additionally, D. desulfuricans strain CSN grew with sulfite but not with thiosulfate, although thiosulfate was disproportionated. Two sulfur-reducing bacteria, four phototrophic sulfur-oxidizing bacteria (incubated in the dark), and Thiobacillus denitrificans did not disproportionate thiosulfate or sulfite. Desulfovibrio sulfodismutans and D. desulfuricans CSN formed sulfate from thiosulfate or sulfite even when simultaneously oxidizing hydrogen or ethanol, or in the presence of 50 mM sulfate. The capacities of sulfate reduction and of thiosulfate and sulfite disproportionation were constitutively present. Enzyme activities required for sulfate reduction (ATP sulfurylase, pyrophosphatase, APS reductase, sulfite reductase, thiosulfate reductase, as well as adenylate kinase and hydrogenase) were detected in sufficient activities to account for the growth rates observed. ADP sulfurylase and sulfite oxidoreductase activities were not detected. Disproportionation was sensitive to the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) but not to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). It is proposed that during thiosulfate and sulfite disproportionation sulfate is formed via APS reductase and ATP sulfurylase, but not by sulfite oxidoreductase. Reversed electron transport must be assumed to explain the reduction of thiosulfate and sulfite by the electrons derived from APS reductase.
128 citations
TL;DR: The isolation of these bacteria indicates that acetogenic bacteria are inhabitants of the rumen of the bovine fed a typical diet and suggests that they may be participants in the utilization of hydrogen in theRumen ecosystem.
Abstract: Five strains of acetogenic bacteria were isolated by selective enrichment from the rumen of a mature Hereford crossbred steer fed a typical high forage diet. Suspensions of rumen bacteria, prepared from contents collected 7 h postfeeding, blended and strained through cheesecloth, were incubated in a minimal medium containing 10% clarified rumen fluid under either H2:CO2 (80:20) or N2:CO2 (80:20) headspace atmosphere. The selection criterion was an increment of acetate in the enrichments incubated under H2:CO2. Periodically, the enrichment broths were plated onto agar media and presumed acetogenic bacteria subsequently were screened for acetate production. Selected acetogenic bacteria utilized a pressurized atmosphere of H2:CO2 to form acetate in quantities 2 to 8-fold higher than when grown under N2:CO2. All presumptive acetogenic isolates were derived from either the 10-7 or 10-8 dilutions of rumen contents. All 5 strains were Gram-positive rods, and all utilized formate, glucose and CO. One strain required, and all were stimulated by, rumen fluid. No spores were observed with phase-contast microscopy and two strains were motile. No methane was detected in the headspace of pure cultures grown under either gas phase. The isolation of these bacteria indicates that acetogenic bacteria are inhabitants of the rumen of the bovine fed a typical diet and suggests that they may be participants in the utilization of hydrogen in the rumen ecosystem. Strain 139B (= ATCC 43876) is named Acetitomaculum ruminis gen. nov., sp. nov. and is the type strain of this new species.
121 citations
TL;DR: From the shallow geothermally heated seafloor at the beach of Porto di Levante (Vulcano, Italy) 8 strains of long, tiny rods were isolated, which represent the first marine metal-mobilizing bacteria, and represent a new group within the genus Thiobacillus.
Abstract: From the shallow geothermally heated seafloor at the beach of Porto di Levante (Vulcano, Italy) 8 strains of long, tiny rods were isolated, which represent the first marine metal-mobilizing bacteria. Cells are Gram negative. They grow in a temperature range between 23 and 41°C with an optimum around 37°C at a salt concentration of up to 6.0% NaCl. The isolates are obligately chemolithotrophic, acidophilic aerobes which use sulfidic ores, elemental sulfur or ferrous iron as energy sources and procedure sulfuric acid. They show an upper pH-limit of growth at around 4.5. The G+C content of their DNA is around 64 mol%. Based on the results of the DNA-DNA hybridization they represent a new group within the genus Thiobacillus. Isolate LM3 is described as the type strain of the new species Thiobacillus prosperus.
117 citations
TL;DR: From a detailed analysis of the restriction properties of the enzymes SAU3 AI, MboI and DpnI, it was concluded that a subset of GATC sequences in P. denitrificans DNA may be methylated at an unusual position and possesses at least one potent host-dependent restriction/modification system which affects conjugation.
Abstract: A selection scheme was devised to isolate Paracoccus denitrificans mutants with increased recipient qualities in transfer experiments, using broad host range plasmids. In some of the mutants obtained, a DNA modifying activity that prevents the activity of the restriction endonucleases BamHI and BglII on isolated P. denitrificans DNA had simultaneously been lost. From a detailed analysis of the restriction properties of the enzymes SAU3 AI, MboI and DpnI, it was concluded that a subset of GATC sequences in P. denitrificans DNA may be methylated at an unusual position. It was concluded that P. denitrificans possesses at least one potent host-dependent restriction/modification system which affects conjugation. In addition to the class of restriction-defective mutants, at least one other class of enhanced transfer mutants with unknown defect(s) was isolated. Strains, in which the two mutant classes were combined, exhibited transfer frequencies which were significantly higher than strains containing either mutation alone. Such double mutant strains appeared to be well suited for future experiments like complementation analysis, transposon mutagenesis and gene replacement by homologous recombination.
TL;DR: Failure of whole cells to oxidize formate, and the absence of formaldehyde-and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS, which suggested that the synthesis of these enzymes is not under coordinate control.
Abstract: The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent Km values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde- and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.
TL;DR: It is concluded that secretion of glutamate in C. glutamicum occurs by a special efflux carrier system, and the specificity and the kinetics of glutamate transport as well as the observed regulation phenomena are concluded.
Abstract: Corynebacterium glutamicum effectively secretes L-glutamate when growing under biotin limitation. The secretion of glutamate was studied with respect to kinetic and energetic parameters: rate of glutamate uptake and efflux, specificity of transport, dependence of efflux on the energy state of the cell, concentration gradient of glutamate and ions, and membrane potential. By comparing these parameters when measured in biotin-limited, i.e. “producer” cells, and biotin-supplemented, i.e. “non-producer” cells, respectively, the following conclusions could be drawn: 1. The efflux of L-glutamate in C. glutamicum cannot be explained by passive permeation of this amino acid through the plasma membrane, as it has been assumed in the generally accepted model of glutamate secretion in biotin-limited cells. 2. It is unlikely that the efflux of glutamate occurs via an inversion of the glutamate uptake system. 3. Based on our results concerning the specificity and the kinetics of glutamate transport as well as the observed regulation phenomena, we conclude that secretion of glutamate in C. glutamicum occurs by a special efflux carrier system.
TL;DR: The pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum was purified to homogeneity and partially characterized, and Ferredoxin from Closridium pasteurianum could be used as physiological electron acceptor.
Abstract: The pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum was purified to homogeneity and partially characterized. A 9.2-fold purification was achieved in a three step purification procedure: ammonium sulfate fractionation, chromatography on Phenyl Sepharose and on Procion Blue H-EGN12. The pure enzyme exhibited a specific activity of 25 U/mg of protein. Homogeneity of the pyruvate-ferredoxin oxidoreductase was confirmed by native polyacrylamide gel electrophoresis and sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight was determined to be 123,000/monomer. The subunit composition of the native enzyme could not be determined because of the instability of the pure enzyme. The pyruvate-ferredoxin oxidoreductase is sensitive to oxygen and dilution during purification. The dilution inactivation could be partially overcome by the addition of 300 microM coenzyme A or 50% ethyleneglycol. A thiamine pyrophosphate content of 0.39 mol per mol of enzyme monomer was found, the iron and sulfur content was 4.23 and 0.91, respectively. The pH-optimum was at pH 7.5 and the temperature optimum was at 60 degrees C. Kinetic constants were measured in the forward reaction. The apparent Km for pyruvate and coenzyme A were 322 microM and 3.7 microM, respectively. With 2-ketobutyrate the pyruvate-ferredoxin oxidoreductase showed 12.5% of the activity compared to pyruvate. No activity was found with 2-ketoglutarate. Ferredoxin from Clostridium pasteurianum could be used as physiological electron acceptor.
TL;DR: From the enzyme outfit it is concluded that in A. fulgidus lactate is oxidized to CO2 via a modified acetyl-CoA/carbon monoxide dehydrogenase pathway involving C1-intermediates otherwise only used by methanogenic bacteria.
Abstract: Archaeoglobus fulgidus is an extremely thermophilic archaebacterium that can grow at the expense of lactate oxidation with sulfate to CO2 and H2S. The organism contains coenzyme F420, tetrahydromethanopterin, and methanofuran which are coenzymes previously thought to be unique for methanogenic bacteria. We report here that the bacterium contains methylenetetrahydromethanopterin: F420 oxidoreductase (20 U/mg), methenyltetrahydromethanopterin cyclohydrolase (0.9 U/mg), formyltetrahydromethanopterin: methanofuran formyltransferase (4.4 U/mg), and formylmethanofuran: benzyl viologen oxidoreductase (35 mU/mg). Besides these enzymes carbon monoxide: methyl viologen oxidoreductase (5 U/mg), pyruvate: methyl viologen oxidoreductase (0.7 U/mg), and membranebound lactate: dimethylnaphthoquinone oxidoreductase (0.1 U/mg) were found. 2-Oxoglutarate dehydrogenase, which is a key enzyme of the citric acid cycle, was not detectable. From the enzyme outfit it is concluded that in A. fulgidus lactate is oxidized to CO2 via a modified acetyl-CoA/carbon monoxide dehydrogenase pathway involving C1-intermediates otherwise only used by methanogenic bacteria.
TL;DR: Polysaccharides excreted by cowpea Rhizobium strains JLn(c) and RA-1 were mixtures of complex acidic exopolysaccharide and low molecular weight neutral glucans and non-carbohydrate substituents were identified by infrared and proton nuclear magnetic resonance spectroscopy as well as by chemical analysis.
Abstract: Polysaccharides excreted by cowpea Rhizobium strains JLn(c) and RA-1 were mixtures of complex acidic exopolysaccharides and low molecular weight neutral glucans. These polymers were fractionated using gel filtration chromatography. Purified fractions of the acidic heteropolymer reacted with peanut agglutinin to give precipitin bands when subjected to Ouchterlony gel diffusion. The acidic exopolysaccharide was found to contain mainly glucose, galactose, glucuronic acid, mannose and fucose. The non-carbohydrate substituents of the acidic heteropolymer were pyruvate, acetate and uronate which were identified by infrared and proton nuclear magnetic resonance spectroscopy as well as by chemical analysis.
TL;DR: The results indicated that probably both, ΔpH and ΔΨ drive sulfate accumulation and that sulfate is taken up electrogenically in symport with more than 2 protons, and not in favour of an ATP-dependent transport system.
Abstract: Uptake of 35S-labelled sulfate was studied with a new isolate of Desulfovibrio desulfuricans, strain CSN. Micromolar additions of sulfate (1–10 μM or nmol/mg protein) to cell suspensions incubated in 150 mM KCl at-1°C were almost completely taken up and accumulated about 5,000-fold. Accumulation was not influenced by incubation in NaCl instead of KCl, by acidic pH (5.5) or by incubation under air for 10 min. In alkaline milieu (pH 8.5), after prolonged contact with air (2 h), or after growth with excess sulfate or thiosulfate as electron acceptor, the amount taken up was diminished approximately by half. Pasteurization inhibited sulfate uptake completely. With increasing concentrations of added sulfate (0.1 to 2.5 mM) the intracellular concentration increased only slowly up to 25 mM, and the accumulation factor decreased down to 8. Sulfate transport was reversible. Accumulated sulfate was rapidly lost from the cells after addition of excess non-labelled sulfate or after addition of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The ATPase inhibitor dicyclohexylcarbodiimide (DCCD) specifically inhibited sulfate reduction but had no immediate influence on sulfate accumulation. Addition of the phosphate analogue arsenate (5 mM) was without effect. These results were not in favour of an ATP-dependent transport system. The K+-H+-antiporter nigericin (in 150 mM KCl) and the Na+-H+-antiporter monensin (in 150 mM NaCl) caused partial inhibition of sulfate accumulation, whereas the K+-transporter valinomycin (in 150 mM KCl) and the Na+-H+ exchange inhibitor amiloride (2 mM) were without effect. The permeant thiocyanate anion (150 mM) inhibited sulfate uptake by 60% at pH 7, and completely at pH 8.5. Although the effects of the different ionophores on the chemiosmotic gradients have not been studied so far, the results indicated that probably both, ΔpH and ΔΨ drive sulfate accumulation and that sulfate is taken up electrogenically in symport with more than 2 protons. The structural sulfate analogues tungstate and molybdate (0.1 mM, each) did not affect sulfate accumulation, although molybdate inhibited sulfate reduction. Chromate completely blocked both of these activities. Sulfite and selenite caused little or no decrease of sulfate accumulation, whereas with thiosulfate and selenate significant inhibition was observed.
TL;DR: The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed and its properties were determined by studying the isotope exchange reaction.
Abstract: Extracts of denitrifying bacteria grown anaerobically with phenol and nitrate catalyzed an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate. This exchange reaction is ascribed to a novel enzyme, phenol carboxylase, initiating the anaerobic degradation of phenol by para-carboxylation to 4-hydroxybenzoate. Some properties of this enzyme were determined by studying the isotope exchange reaction. Phenol carboxylase was rapidly inactivated by oxygen; strictly anoxic conditions were essential for preserving enzyme activity. The exchange reaction specifically was catalyzed with 4-hydroxybenzoate but not with other aromatic acids. Only the carboxyl group was exchanged; [U-14C]phenol was not exchanged with the aromatic ring of 4-hydroxybenzoate. Exchange activity depended on Mn2+ and inorganic phosphate and was not inhibited by avidin. Ortho-phosphate could not be substituted by organic phosphates nor by inorganic anions; arsenate had no effect. The pH optimum was between pH 6.5–7.0. The specific activity was 100 nmol 14CO2 exchange · min-1 · mg-1 protein. Phenol grown cells contained 4-hydroxybenzoyl CoA synthetase activity (40 nmol · min-1 · mg-1 protein). The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed.
TL;DR: Cell extracts of acetate grown Methanosarcina barkeri indicate that the archaebacterium contains the enzyme carbonic anhydrase (EC 4.2.1).
Abstract: Cell extracts (27000xg supernatant) of acetate grown Methanosarcina barkeri were found to have carbonic anhydrase activity (0.41 U/mg protein), which was lost upon heating or incubation with proteinase K. The activity was inhibited by Diamox (apparent K
i=0.5 mM), by azide (apparent K
i=1 mM), and by cyanide (apparent K
i=0.02 mM). These and other properties indicate that the archaebacterium contains the enzyme carbonic anhydrase (EC 4.2.1.1). Evidence is presented that the protein is probably located in the cytoplasm. Methanol or H2/CO2 grown cells of M. barkeri showed no or only very little carbonic anhydrase activity. After transfer of these cells to acetate medium the activity was “induced” suggesting a function of this enzyme in acetate fermentation to CO2 and CH4. Interestingly, Desulfobacter postgatei and Desulfotomaculum acetoxidans, which oxidize acetate to 2 CO2 with sulfate as electron acceptor, were also found to exhibit carbonic anhydrase activity (0.2 U/mg protein).
TL;DR: A new family, the Methanocorpusculaceae fam.
Abstract: Two new methanogenic bacteria, Methanocorpusculum sinense spec. nov. strain DSM 4274 from a pilot plant for treatment of distillery wastewater in Chengdu (Province Sichuan, China), and Methanocorpusculum bavaricum spec. nov. strain DSM 4179, from a wastewater pond of the sugar factory in Regensburg (Bavaria, FRG) are described. Methanocorpusculum strains are weakly motile and form irregularly coccoid cells, about 1 micron in diameter. The cell envelope consists of a cytoplasmic membrane and a S-layer, composed of hexagonally arranged glycoprotein subunits with molecular weights of 90,000 (Methanocorpusculum parvum), 92,000 (M. sinense), and 94,000 (M. bavaricum). The center-to-center spacings are 14.3 nm, 15.8 nm and 16.0 nm, respectively. Optimal growth of strains is obtained in the mesophilic temperature range and at a pH around 7. Methane is produced from H2/CO2, formate, 2-propanol/CO2 and 2-butanol/CO2 by M. parvum and M. bavaricum, whereas M. sinense can only utilize H2/CO2 and formate. Growth of M. sinense and M. bavaricum is dependent on the presence of clarified rumen fluid. The G + C content of the DNA of the three strains is ranging from 47.7-53.6 mol% as determined by different methods. A similar, but distinct polar lipid pattern indicates a close relationship between the three Methanocorpusculum species. The polyamine patterns of M. parvum, M. sinense and M. bavaricum are similar, but distinct from those of other methanogens and are characterized by a high concentration of the otherwise rare 1,3-diaminopropane. Quantitative comparison of the antigenic fingerprint of members of Methanocorpusculum revealed no antigenic relationship with any one of the reference methanogens tested. On the basis of the distant phylogenetic position of M. parvum and the data presented in this paper a new family, the Methanocorpusculaceae fam. nov., is defined.
TL;DR: Growth experiments mostly revealed diauxic substrate utilization pattern using different combinations of glycine, sarcosine, and betaine (plus formate) and inocula from different precultures, where Glycine was always utilized first, what coincided with the presence of glycinesine reductase activity under all growth conditions except for serine as substrate.
Abstract: The obligate anaerobe Eubacterium acidaminophilum metabolized the glycine derivatives sarcosine (N-monomethyl glycine) and betaine (N-trimethyl glycine) only by reduction in a reaction analogous to glycine reductase. Using formate as electron donor, sarcosine and betaine were stoichiometrically reduced to acetate and methylamine or trimethylamine, respectively. The N-methyl groups of the cosubstrates or of the amines produced were not transformed to CO2 or acetate. Under optimum conditions (formate/acceptor ratio of 1 to 1.2, 34°C, pH 7.3) the doubling times were 4.2 h on formate/sarcosine and 3.6 h on formate/betaine. The molar growth yields were 8.15 and 8.5 g dry cell mass per mol sarcosine and betaine, respectively. The assays for sarcosine reductase and betaine reductase were optimized in cell extracts; NADPH was preferred as physiological electron donor compared to NADH, dithioerythritol was used as artificial donor; no requirements for AMP and ADP could be detected. Growth experiments mostly revealed diauxic substrate utilization pattern using different combinations of glycine, sarcosine, and betaine (plus formate) and inocula from different precultures. Glycine was always utilized first, what coincided with the presence of glycine reductase activity under all growth conditions except for serine as substrate. Sarcosine reductase and betaine reductase were only induced when E. acidaminophilum was grown on sarcosine and betaine, respectively. Creatine was metabolized via sarcosine. [75Se]-selenite labeling revealed about the same pattern of predominant labeled proteins in glycine-, sarcosine-, and betaine-grown cells.
TL;DR: A new strictly anaerobic bacterium was isolated from an enrichment culture with glutarate as sole substrate and freshwater sediment as inoculum, however, glutarate was not metabolized by the pure culture, and was described as a new species of the genus Sporomusa, S. malonica.
Abstract: A new strictly anaerobic bacterium was isolated from an enrichment culture with glutarate as sole substrate and freshwater sediment as inoculum, however, glutarate was not metabolized by the pure culture. The isolate was a mesophilic, spore-forming, Gram-negative, motile curved rod. It fermented various organic acids, alcohols, fructose, acetoin, and H2/CO2 to acetate, usually as the only product. Other acids were fermented to acetate and propionate or acetate and butyrate. Succinate and malonate were decarboxylated to propionate or acetate, respectively, and served as sole sources of carbon and energy for growth. No inorganic electron acceptors except CO2 were reduced. Yeast extract (0.05% w/v) was required for growth. Small amounts of cytochrome b were detected in membrane fractions. The guanine-plus-cytosine content of the DNA was 44.1±2 mol%. The isolate is described as a new species of the genus Sporomusa, S. malonica.
TL;DR: It is proposed that the physiological function of hydrogen production is the introduction of protons as terminal electron acceptors, which might give rise to continuation of the pyruvate decarboxylation and consequently of the acetate formation, thereby increasing the efficiency of fermentative energy generation.
Abstract: The hydrogenase-catalyzed hydrogen production exhibited by the unicellular cyanobacterium Cyanothece 7822 during anoxic incubation in the dark is a result of the fermentative degradation of carbon reserves. Simultaneously with hydrogen production, evolution of carbon dioxide was detected, and excretion of ethanol, lactate, formate and acetate was demonstrated. The fermentation balance indicates that carbohydrates are fermented via a branched pathway, in which both the pentose phosphate pathway and glycolysis appear to be involved. It is proposed that the physiological function of hydrogen production is the introduction of protons as terminal electron acceptors. This removal of reducing equivalents might give rise to continuation of the pyruvate decarboxylation and consequently of the acetate formation, thereby increasing the efficiency of fermentative energy generation.
TL;DR: It is concluded, that uptake of branched-chain amino acid transport proceeds via a secondary active Na+-coupled symport mechanism.
Abstract: The transport of branched-chain amino acids was characterized in intact cells of Corynebacterium glutamicum ATCC 13032. Uptake and accumulation of these amino acids occur via a common specific carrier with slightly different affiniteis for each substrate (Km[Ile]=5.4 μM, Km[Leu]=9.0 μM, Km[Val]=9.5 μM). The maximal uptake rates for all three substrates were very similar (0.94–1.30 nmol/mg dw · min). The optimum of amino acid uptake was at pH 8.5 and the activation energy was determined to be 80 kJ/mol. The transport activity showed a marked dependence on the presence of Na+ ions and on the membrane potential, but was independent of an existing proton gradient. It is concluded, that uptake of branched-chain amino acid transport proceeds via a secondary active Na+-coupled symport mechanism.
TL;DR: From the frequency of isolation, it appears that extremely thermophilic methanococci are the predominant representatives of the methanogenic archaebacteria occurring at deep sea hydrothermal vents.
Abstract: An extremely thermophilic methanogen was isolated from hydrothermal vent sediment (80°–120° C) collected from the Guaymas Basin, Gulf of California, at a depth of approximately 2000 m. The isolate was a characteristic member of the genus Methanococcus based on its coccoid morphology, ability to produce methane from CO2 and H2, and DNA base composition (31.4 mol% G+C); it is distinguished from previously described extremely thermophilic vent methanogens by its ability to grow and produce methane from formate and in the composition of membrane lipids. The temperature range for growth was 48°–94° C (optimum near 85° C); the pH optimum was 6.0. The isolate grew autotrophically but was stimulated by selenium and growth nutrients supplied by yeast extract and trypticase. Extracted polar lipids consisted primarily of diphytanyl glycerol diether (62%), macrocyclic glycerol diether (15.3%), and dibiphytanyl glycerol tetraether (11.8%). Neutral lipids were dominated by a series of C30 isoprenoids; in addition, a novel series of C35 isoprenoids were detected. The isolate appears to be a close relative of the previously described Methanococcus jannaschii, isolated from the East Pacific Rise hydrothermal vent system. From the frequency of isolation, it appears that extremely thermophilic methanococci are the predominant representatives of the methanogenic archaebacteria occurring at deep sea hydrothermal vents.
TL;DR: The transposon-containing streptococcal plasmids pAM211, pCF10, and pINY1275 have been transferred to the Gram-positive anaerobe Clostridium acetobutylicum and used as donors in mating experiments with tetracycline-sensitive Bacillus subtilis and Streptococcus lactis subspec.
Abstract: The transposon-containing streptococcal plasmids pAM211, pCF10, and pINY1275 have been transferred at high frequency (10-2–10-3 per recipient, selecting for tetracycline resistance) to the Gram-positive anaerobe Clostridium acetobutylicum. Selection in the presence of two antibiotics (tetracycline and erythromycin) with the plasmids pAM 180 and pINY1275 yielded only low numbers of transconjugants (≤10-8 per recipient). Matings were done by combining liquid and filter mating procedures under anaerobic conditions. No plasmid DNA could be detected in the transconjugants selected on a minimal medium in the presence of tetracycline. DNA-DNA hybridization experiments with restricted chromosomal DNA using biotinylated pAM120::Tn916 as probe revealed the presence of homologous sequences in the transconjugants but not in Clostridium acetobutylicum wild type. The transconjugants were used as donors in mating experiments with tetracycline-sensitive Bacillus subtilis and Streptococcus lactis subspec. diacetylactis. In both cases tetracycline-resistant strains were found. Transfer frequencies in these experiments were less than 10-7 per recipient.
TL;DR: The results indicate that in acid soils nitrification by ureolytic ammonium-oxidizing chemolithotrophs may not be restricted to microsites of neutral pH.
Abstract: An ureolytic ammonium-oxidizing chemolithotroph belonging to the genus Nitrosospira was shown to nitrify at pH 4.5 in a pH-stat with urea as a substrate. With ammonium as the sole substrate nitrification did not occur at pH values below 5.5. Nitrosomonas europaea ATCC 19718 and Nitrosospira briensis ATCC 25971 did not possess urease activity. The results indicate that in acid soils nitrification by ureolytic ammonium-oxidizing chemolithotrophs may not be restricted to microsites of neutral pH.
TL;DR: The initial reactions possibly involved in the acrobic and anaerobic metabolism of aromatic acids by a denitrifying Pseudomonas strain were studied and several acyl CoA synthetases were found supporting the view that activation of several aromatic acids preceeds degradation.
Abstract: The initial reactions possibly involved in the acrobic and anaerobic metabolism of aromatic acids by a denitrifying Pseudomonas strain were studied. Several acyl CoA synthetases were found supporting the view that activation of several aromatic acids preceeds degradation. A benzoyl CoA synthetase activity (AMP forming) (apparent K
m values of the enzyme from nitrate grown cells: 0.01 mM benzoate, 0.2 mM ATP, 0.2 mM coenzyme A) was present in aerobically grown and anaerobically, nitrate grown cells when benzoate or other aromatic acids were present. In addition to benzoate and fluorobenzoates, also 2-amino-benzoate was activated, albeit with unfavorable K
m (0.5 mM 2-aminobenzoate). A 2-aminobenzoyl CoA synthetase (AMP forming) was induced both aerobically and anaerobically with 2-aminobenzoate as growth substrate which had a similar substrate spectrum but a low K
m for 2-aminobenzoate (<0.02 mM). Anaerobic growth on 4-hydroxybenzoate induced a 4-hydroxybenzoyl CoA synthetase, and cyclohexanecarboxylate induced another synthetase. In contrast, 3-hydroxybenzoate and phenyl-acetate grown anaerobic cells appeared not to activate the respective substrates at sufficient rates. Contrary to an earlier report extracts from aerobic and anaerobic 2-aminobenzoate grown cells catalysed a 2-aminobenzoyl CoA-dependent NADH oxidation. This activity was 10–20 times higher in aerobic cells and appeared to be induced by 2-aminobenzoate and oxygen. In vitro, 2-aminobenzoyl CoA reduction was dependent on 2-aminobenzoyl CoA NAD(P)H, and oxygen. A novel mechanism of aerobic 2-aminobenzoate degradation is suggested, which proceeds via 2-aminobenzoyl CoA.
TL;DR: A new strain of the green sulfur bacteria was isolated from the monimolimnion of Buchensee (near Radolfzell, Lake Constance region, FRG) and represents a new species, P. Phaeoclathratiforme sp.
Abstract: A new strain of the green sulfur bacteria was isolated from the monimolimnion of Buchensee (near Radolfzell, Lake Constance region, FRG). Single cells were rod-shaped, nonmotile and contained gas vacuoles. Typical net-like colonies were formed by ternary fission of the cells. As photosynthetic pigments bacteriochlorophylls a, e, isorenieratene and β-isorenieratene were present. Sulfide, sulfur and thiosulfate were used as electron donors during anaerobic phototrophic growth. Besides carbon dioxide, acetate and propionate could serve as carbon sources under mixotrophic conditions in the light. Like all other members of the green sulfur bacteria, the new bacterium is strictly anaerobic and obligately phototrophic. The possession of gas vacuoles and the formation of net-like colonies and the guanine plus cytosine content of the DNA (47.9 mol% G+C) are typical characteristics of the genus Pelodictyon. Because of its photosynthetic pigments which differ from those of Pelodictyon clathratiforme, strain BU 1 represents a new species, P. Phaeoclathratiforme sp. nov.
TL;DR: In this paper, it is suggested that the superoxide radicals arise from semiquinone radicals which are formed by one electron reduction of quinone in a Na+-independent reaction sequence and then dismutate in an Na+ and HQNO sensitive reaction to quinones and quinol.
Abstract: Membranes of Klebsiella pneumoniae, grown anaerobically on citrate, contain a NADH oxidase activity that is activated specifically by Na+ or Li+ ions and effectively inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Cytochromes b and d were present in the membranes, and the steady state reduction level of cytochrome b increased on NaCl addition. Inverted bacterial membrane vesicles accumulated Na+ ions upon NADH oxidation. Na+ uptake was completely inhibited by monensin and by HQNO and slightly stimulated by carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), thus indicating the operation of a primary Na+ pump. A Triton extract of the bacterial membranes did not catalyze NADH oxidation by O2, but by ferricyanide or menadione in a Na+-independent manner. The Na+-dependent NADH oxidation by O2 was restored by adding ubiquinone-1 in micromolar concentrations. After inhibition of the terminal oxidase with KCN, ubiquinol was formed from ubiquinone-1 and NADH. The reaction was stimulated about 6-fold by 10 mM NaCl and was severely inhibited by low amounts of HQNO. Superoxide radicals were formed during electron transfer from NADH to ubiquinone-1. These radicals disappeared by adding NaCl, but not with NaCl and HQNO. It is suggested that the superoxide radicals arise from semiquinone radicals which are formed by one electron reduction of quinone in a Na+-independent reaction sequence and then dismutate in a Na+ and HQNO sensitive reaction to quinone and quinol. The mechanism of the respiratory Na+ pump of K. pneumoniae appears to be quite similar to that of Vibrio alginolyticus.