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G Gottschalk

Publications and source records attributed to G Gottschalk.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cloning, sequencing and expression of the genes encoding the sodium translocating N5-methyltetrahydromethanopterin : coenzyme M methyltransferase of the methylotrophic archaeon Methanosarcina mazei Gö1.

The N5-methyltetrahydromethanopterin:coenzyme M methyltransferase of Methanosarcina mazei Gö1 is a membrane-associated, corrinoid-containing protein that uses a transmethylation reaction to drive an energy-conserving sodium ion pump. The eight open reading frames encoding the eight different subunits of the methyltransferase were identified and sequenced. All of these subunits are shown to be heterologously expressed in minicells of the Escherichia coli mutant DK6. Sequence comparisons with the methyltransferases of thermophilic and hypothermophilic methanogenic archaea are presented. The participation of the gene product of mtrD in sodium ion translocation as well as a consensus sequence of a corrinoid binding motif in MtrA are discussed.

Amino Acid Sequence↗
PubMed1998

Biochemistry of coenzyme B12-dependent glycerol and diol dehydratases and organization of the encoding genes.

Glycerol and diol dehydratases exhibit a subunit composition of alpha 2 beta 2 gamma 2 and contain coenzyme B12 in the base-on form. The dehydratase reaction proceeds via a radical mechanism. The dehydratases are subject to reaction inactivation by the substrate glycerol which is caused by a cessation of the catalytic cycle because coenzyme B12 is not regenerated, instead 5'-deoxyadenosine and a catalytically inactive cobalamin are formed. The genetic organization of the dehydratase genes is quite similar in all organisms. Downstream of the dehydratase genes an open reading frame encoding a polypeptide of approximately 600 amino acids was identified which is apparently involved in the reactivation of suicide-inactivated enzyme.

Amino Acid Sequence↗
PubMed1998

Construction and use of an ipb DNA module to generate Pseudomonas strains with constitutive trichloroethene and isopropylbenzene oxidation activity.

Pseudomonas sp. strain JR1 exhibits trichloroethene (TCE) oxidation activity with isopropylbenzene (IPB) as the inducer substrate. We previously reported the genes encoding the first three enzymes of the IPB-degradative pathway (ipbA1, ipbA2, ipbA3, ipbA4, ipbB, and ipbC) and identified the initial IPB dioxygenase (IpbA1 A2A3A4) as responsible for TCE cooxidation (U. Pflugmacher, B. Averhoff, and G. Gottschalk, Appl. Environ. Microbiol. 62:3967-3977, 1996). Primer extension analyses revealed multiple transcriptional start points located upstream of the translational initiation codon of ipbA1. The transcription from these start sites was found to be IPB dependent. Thirty-one base pairs upstream of the first transcriptional start point tandemly repeated DNA sequences overlapping the -35 region of a putative sigma 70 promoter were found. These repeats exhibit significant sequence similarity to the operator-promoter region of the xyl meta operon in Pseudomonas putida, which is required for the binding of XylS, a regulatory protein of the XylS (also called AraC) family. These similarities suggest that the transcription of the IPB dioxygenase genes is modulated by a regulatory protein of the XylS/AraC family. The construction of an ipb DNA module devoid of this ipb operator-promoter region and the stable insertion of this DNA module into the genomes of different Pseudomonas strains resulted in pseudomonads with constitutive IPB and TCE oxidation activities. Constitutive TCE oxidation of two such Pseudomonas hybrid strains, JR1A::ipb and CBS-3::ipb, was found to be stable for more than 120 generations in antibiotic-free medium. Evaluation of constitutive TCE degradation rates revealed that continuous cultivation of strain JR1A::ipb resulted in a significant increase in rates of TCE degradation.

Benzene Derivatives↗
PubMed1997

Glycerol conversion to 1,3-propanediol by Clostridium pasteurianum: cloning and expression of the gene encoding 1,3-propanediol dehydrogenase.

When grown on glycerol as sole carbon and energy source, cell extracts of Clostridium pasteurianum exhibited activities of glycerol dehydrogenase, dihydroxyacetone kinase, glycerol dehydratase and 1,3-propanediol dehydrogenase. The genes encoding the latter two enzymes were cloned by colony hybridization using the dhaT gene of Citrobacter freundii as a heterologous DNA probe and expressed in Escherichia coli. The native molecular mass of 1,3-propanediol dehydrogenase (DhaT) is 440,000 Da. The dhaT gene of C. pasteurianum was subcloned and its nucleotide sequence (1158 bp) was determined. The deduced gene product (41,776 Da) revealed high similarity to DhaT of C. freundii (80.5% identity; 89.8% similarity).

Alcohol Dehydrogenase↗
PubMed1997

Biosynthesis of poly(4-hydroxybutyric acid) by recombinant strains of Escherichia coli.

The aim of this study was the production of the homopolyester poly(4-hydroxybutyric acid) (poly(4HB)) with recombinant strains of Escherichia coli. Wild-type strains and other widely used non-recombinant strains of E. coli are not able to produce polyhydroxyalkanoic acids (PHA) as storage compounds and cannot utilize 4-hydroxybutyric acid as sole carbon source. Accordingly, hybrid plasmids of pBluescript vectors were constructed which harbored the Alcaligenes eutrophus PHA synthase gene (phaC) and the Clostridium kluyveri orfZ putatively encoding a 4-hydroxybutyric acid-coenzyme A transferase. A 3.5-kb genomic SmaI/ApaI fragment from A. eutrophus, which comprises phaC, and a 1.8-kb genomic ApaI/EcoRI fragment from C kluyveri, which contained orfZ, were inserted into the SmaI and EcoRI sites of the vectors pKS- and pSK-, respectively. The two resulting plasmids pSKSE5.3 and pKSSE5.3 comprising phaC and orfZ colinear or antilinear to lacZ, respectively, were transformed into E. coli XL1-Blue. Recombinant strains synthesized the homopolyester poly(4HB), when the cells were cultivated in Luria-Bertani broth and if glucose and 4-hydroxybutyric acid were provided as carbon sources. If glucose was omitted, a copolyester of 3-hydroxybutyric acid and 4-hydroxybutyric acid was accumulated. The homopolyester poly(4HB) was also accumulated during cultivation of these strains in M9 mineral salts medium containing glucose plus 4-hydroxybutyric acid as carbon sources. Poly(4HB) could amount up to approximately 80% (w/w) of the cell dry matter if E. coli XL1-Blue harboring pKSSE5.3 was cultivated in M9 mineral salts medium and if the cultures were not sufficiently supplied with oxygen. 4HB was also incorporated into PHA if gamma-butyrolactone was used as carbon source. If levulinic acid, 4-hydroxyvaleric acid or gamma-valerolactone were used as carbon sources, only very low amounts of PHA were accumulated which did not contain 4-hydroxyalkanoic acids as constituents.

4-Butyrolactone↗
PubMed1996

Sodium ion translocation by N5-methyltetrahydromethanopterin: coenzyme M methyltransferase from Methanosarcina mazei Gö1 reconstituted in ether lipid liposomes.

The N5-methyltetrahydromethanopterin (H,MPT):coenzyme M methyltransferase is a membrane associated, corrinoid-containing protein that uses the methylation of coenzyme M (HS-CoM) by methyl-tetrahydromethanopterin to drive an energy-conserving sodium ion pump. The enzyme was purified from acetate-grown Methanosarcina mazei Gö1 by a two-step solubilization with n-octyl-beta-glucoside, chromatography on hydroxyapatite, and by gel filtration on Superdex 200 or Sepharose CL-6B. The highly purified protein was apparently composed of six different subunits of 34, 28, 20, 13, 12, and 9 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native enzyme exhibited an apparent molecular mass of about 380 kDa. During purification, the enzyme was stabilized with 10 microM hydroxocobalamin. The highest specific activity reached during purification was 10.4 U/mg. The purified enzyme was reconstituted in monolayer liposomes prepared from ether lipids of M. mazei Gö1. In experiments with radioactive sodium ions, it was shown that the methyltransferase catalyzes the vectorial translocation of sodium ions across the membrane. Methyltransferase activity was stimulated by sodium ions. 1.7 mol Na-/mol methyl groups transferred were translocated. Methyltetrahydrofolate and methyl-cobalamin could substitute for methyl-H,MPT.

Amino Acid Sequence↗
PubMed1996

Studies on the isopropylbenzene 2,3-dioxygenase and the 3-isopropylcatechol 2,3-dioxygenase genes encoded by the linear plasmid of Rhodococcus erythropolis BD2.

The enzymes responsible for the degradation of isopropylbenzene (IPB) and co-oxidation of trichloroethene (TCE) by Rhodococcus erythropolis BD2 are encoded by the linear plasmid pBD2. Fragments containing IPB catabolic genes were cloned from pBD2 and the nucleotide sequence was determined. By means of database searches and expression of the cloned genes in recombinant strains, we identified five clustered genes, ipbA1A2A3A4C, which encode the three components of the IPB 2,3-dioxygenase system, reductaseIPB (ipbA4), ferredoxinIPB (ipbA3) and the two subunits of the terminal dioxygenase (ipbA1A2), as well as the 3-isopropylcatechol (IPC) 2,3-dioxygenase (ipbC). The protein sequences deduced from the ipbA1A2A3A4C gene cluster exhibited significant homology with the corresponding proteins of analogous degradative pathways in Gram-negative and Gram-positive bacteria, but the gene order differed from most of them. IPB 2,3-dioxygenase and 3-IPC 2,3-dioxygenase could both be expressed in Escherichia coli, but the IPB 2,3-dioxygenase activities were too low to be detected by polarographic and TCE degradative means. However, inhibitor studies with the R. erythropolis BD2 wild-type are in accordance with the involvement of the IPB 2,3-dioxygenase in TCE oxidation.

Amino Acid Sequence↗
PubMed1996

Cloning, sequencing, and expression of isopropylbenzene degradation genes from Pseudomonas sp. strain JR1: identification of isopropylbenzene dioxygenase that mediates trichloroethene oxidation.

Pseudomonas sp. strain JR1, recently isolated with isopropylbenzene (IPB) as the inducer substrate for trichloroethene (TCE) oxidation (B. Dabrock, J. Riedel, J. Bertram, and G. Gottschalk, Arch. Microbiol 158:9-13, 1992), is able to degrade IPB via the meta-cleavage pathway. The genes encoding the first three enzymes in the catabolism of isopropylbenzene were isolated from a genomic library with the broad-host-range cosmid vector pWE15. A 7.6-kb fragment from a 37.7-kb primary cosmid clone was subcloned and sequenced. It contained seven complete open reading frames, designated ipbA1A2orf3A3A4BC. ipbA codes for the three subunits of a multicomponent IPB dioxygenase, ipbB codes for 2,3-dihydro-2,3-dihydroxy-IPB dehydrogenase, and ipbC codes for 3-isopropylcatechol 2,3-dioxygenase. The deduced amino acid sequences of ipbA1A2A3A4BC exhibited the highest homologies with the corresponding proteins of biphenyl-degradative pathways in gram-negative and gram-positive bacteria. The gene products of the ipb genes were identified by an in vitro transcription-translation system on the basis of their expected molecular masses. IPB dioxygenase and 3-isopropylcatechol 2,3-dioxygenase expressed in E. coli oxidized a wide range of alkyl aromatic compounds. Incubation of E. coli cells carrying ipbA1A2A3A4 with IPB and 10O2 yielded reaction products containing both atoms of molecular oxygen, which is in accordance with a dioxygenation reaction. E. coli recombinants harboring and expressing the IPB dioxygenase exhibited the ability to degrade TCE. The ipbA1A2A3A4-carrying E. coli strain required neither IPB nor isopropyl-beta-D-thiogalactopyranoside for induction; the rate of TCE degradation was comparable to that by fully induced Pseudomonas strain JR1.

Amino Acid Sequence↗
PubMed1996

Cloning, sequencing, and overexpression of the genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii.

The genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii were cloned and overexpressed in Escherichia coli. The B12-free enzyme was purified to homogeneity. It consists of three types of subunits whose N-terminal sequences are in accordance with those deduced from the open reading frames dhaB, dhaC, and dhaE, coding for subunits of 60,433 (alpha), 21,487 (beta), and 16,121 (gamma) Da, respectively. The enzyme complex has the composition alpha2beta2gamma2. Amino acid alignments with the subunits of the recently sequenced diol dehydratase of Klebsiella oxytoca (T. Tobimatsu, T. Hara, M. Sakaguchi, Y. Kishimoto, Y. Wada, M. Isoda, T. Sakai, and T. Toraya, J. Biol. Chem. 270:7142-7148, 1995) revealed identities between 51.8 and 70.9%.

Bacterial Proteins↗
PubMed1996

Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri.

A region of genomic DNA from Clostridium kluyveri was cloned in Escherichia coli by a screening strategy which was based on heterologous expression of the clostridial 4-hydroxybutyrate dehydrogenase gene. The gene region (6,575 bp) contained several open reading frames which encoded the coenzyme A (CoA)- and NADP+-dependent succinate-semialdehyde dehydrogenase (sucD), the 4-hydroxybutyrate dehydrogenase (4hbD), and a succinyl-CoA;CoA transferase (cat1), as analyzed by heterologous expression in E. coli. An open reading frame encoding a putative membrane protein (orfY) and the 5' region of a gene encoding a sigma 54-homologous sigma factor (sigL) were identified as well. Transcription was investigated by Northern (RNA) blot analysis. Protein sequence comparisons of SucD and 4HbD revealed similarities to the adhE (aad) gene products from E. coli and Clostridium acetobutylicum and to enzymes of the novel class (III) of alcohol dehydrogenases. A comparison of CoA-dependent aldehyde dehydrogenases is presented.

Aldehyde Oxidoreductases↗
PubMed1995

Analysis of the vhoGAC and vhtGAC operons from Methanosarcina mazei strain Gö1, both encoding a membrane-bound hydrogenase and a cytochrome b.

DNA encompassing the structural genes of two membrane-bound hydrogenases from Methanosarcina mazei Gö1 was cloned and sequenced. The genes, arranged in the order vhoG and vhoA as well as vhtG and vhtA, were identified as those encoding the small and the large subunits of the NiFe hydrogenases [Deppenmeier, U., Blaut, M., Schmidt, B. & Gottschalk, G. (1992) Arch. Microbiol. 157, 505-511]. Northern-blot analysis revealed that the structural genes formed part of two operons, both containing one additional open reading frame (vhoC and vhtC) which codes for a cytochrome b. This conclusion was drawn from the homology of the deduced N-terminal amino acid sequences of vhoC and vhtC and the N-terminus of a 27-kDa cytochrome isolated from Ms. mazei C16. VhoC and VhtC contain four tentative hydrophobic segments which might span the cytoplasmic membrane. Hydropathy plots suggest that His23 and His50 are involved in heme coordination. The comparison of the sequencing data of vhoG and vhtG with the experimentally determined N-terminus of the small subunit indicate the presence of a 48-amino-acid leader peptide in front of the polypeptides. VhoA and VhtA contained the conserved sequence DPCXXC in the C-terminal region, which excludes the presence of a selenocysteine residue in these hydrogenases. Promoter sequences were found upstream of vhoG and vhtG, respectively. Downstream of vhoC, a putative terminator sequence was identified. Alignments of the deduced amino acid sequences of the gene clusters vhoGAC and vhtGAC showed 92-97% identity. Only the C-termini of VhoC and VhtC were not similar.

Amino Acid Sequence↗
PubMed1995

Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii.

Glycerol dehydrogenase (EC 1.1.1.6) and dihydroxyacetone kinase (EC 2.7.1.29) were purified from Citrobacter freundii. The dehydrogenase is a hexamer of a polypeptide of 43,000 Da. The enzyme exhibited a rather broad substrate specificity, but glycerol was the preferred substrate in the physiological direction. The apparent Kms of the enzyme for glycerol and NAD+ were 1.27 mM and 57 microM, respectively. The kinase is a dimer of a polypeptide of 57,000 Da. The enzyme was highly specific for the substrates dihydroxyacetone and ATP; the apparent Kms were 30 and 70 microM, respectively. The DNA region which contained the genes encoding glycerol dehydrogenase (dhaD) and dihydroxyacetone kinase (dhaK) was cloned and sequenced. Both genes were identified by N-terminal sequence comparison. The deduced dhaD gene product (365 amino acids) exhibited high degrees of homology to glycerol dehydrogenases from other organisms and less homology to type III alcohol dehydrogenases, whereas the dhaK gene product (552 amino acids) revealed no significant homology to any other protein in the databases. A large gene (dhaR) of 1,929 bp was found downstream from dhaD. The deduced gene product (641 amino acids) showed significant similarities to members of the sigma 54 bacterial enhancer-binding protein family.

Amino Acid Sequence↗
PubMed1995

Purification of 1,3-propanediol dehydrogenase from Citrobacter freundii and cloning, sequencing, and overexpression of the corresponding gene in Escherichia coli.

1,3-Propanediol dehydrogenase (EC 1.1.1.202) was purified to homogeneity from Citrobacter freundii grown anaerobically on glycerol in continuous culture. The enzyme is an octamer of a polypeptide of 43,400 Da. When tested as a dehydrogenase, the enzyme was most active with substrates containing two primary alcohol groups separated by one or two carbon atoms. In the physiological direction, 3-hydroxypropionaldehyde was the preferred substrate. The apparent Km values of the enzyme for 3-hydroxypropionaldehyde and NADH were 140 and 33 microM, respectively. The enzyme was inhibited by chelators of divalent cations but could be reactivated by the addition of Fe2+. The dhaT gene, encoding the 1,3-propanediol dehydrogenase, was cloned, and its nucleotide sequence (1,164 bp) was determined. The deduced dhaT gene product (387 amino acids, 41,324 Da) showed a high level of similarity to a novel family (type III) of alcohol dehydrogenases. The dhaT gene was overexpressed in Escherichia coli 274-fold by using the T7 RNA polymerase/promoter system.

Alcohol Dehydrogenase↗
PubMed1995

Electron paramagnetic resonance spectroscopic and electrochemical characterization of the partially purified N5-methyltetrahydromethanopterin:coenzyme M methyltransferase from Methanosarcina mazei Gö1.

The N5-methyltetrahydromethanopterin:coenzyme M methyltransferase is a membrane-bound cobalamin-containing protein of Methanosarcina mazei Gö1 that couples the methylation of coenzyme M by methyltetra-hydrosarcinopterin to the translocation of Na+ across the cell membrane (B. Becher, V. Müller, and G. Gottschalk, J. Bacteriol. 174:7656-7660, 1992). We have partially purified this enzyme and shown that, in addition to the cobamide, at least one iron-sulfur cluster is essential for the transmethylation reaction. The membrane fraction or the partly purified protein contains a "base-on" cobamide with a standard reduction potential (Eo') for the Co2+/1+ couple of -426 mV. The iron-sulfur cluster appears to be a [4Fe-4S]2+/1+ type with an Eo' value of -215 mV. We have determined the methyltransferase activity at various controlled redox potentials and demonstrated that the enzyme activity is activated by a one-electron reduction with half-maximum activity occurring at -235 mV in the presence of ATP and -450 mV in its absence. No activation was observed when ATP was replaced by other nucleoside triphosphates or nonhydrolyzable ATP analogs.

Adenosine Triphosphate↗
PubMed1994

Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase.

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m = 59 kDa/subunit), containing 2 +/- 0.2 mol FAD, 13.6 +/- 0.8 mol Fe and 10.8 +/- 1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.

Amino Acid Sequence↗
PubMed1994

Inhibition of the lignin peroxidase of Phanerochaete chrysosporium by hydroxylamino-dinitrotoluene, an early intermediate in the degradation of 2,4,6-trinitrotoluene.

The ability of the white rot fungus Phanerochaete chrysosporium to mineralize 2,4,6-trinitrotoluene (TNT) was studied in the concentration range of 0.36 to 20.36 mg/liter. The initial rate of 14CO2 formation was 30% in 4 days at 0.36 mg of [14C]TNT per liter and decreased to 5% in 4 days at 20.36 mg of [14C]TNT per liter. Such a pronounced inhibition was not observed when a mixture of [14C]2-amino-4,6-dinitrotoluene and [14C]4-amino-2,6-dinitrotoluene was used as a substrate. 2-Hydroxylamino-4,6-dinitrotoluene and its isomer 4-hydroxylamino-2,6-dinitrotoluene were identified as the first detectable degradation products of TNT. Their transient accumulation correlated with the inhibition of TNT degradation and of the veratryl alcohol oxidase activity of lignin peroxidase. With purified lignin peroxidase H8, it could be shown that the two isomers of hydroxylamino-dinitrotoluene were oxidized by lignin peroxidase. The corresponding nitroso-dinitrotoluenes apparently were formed, as indicated by the formation of azoxy-tetranitrotoluenes.

Alcohol Oxidoreductases↗
PubMed1994

Identification and characterization of a transmissible linear plasmid from Rhodococcus erythropolis BD2 that encodes isopropylbenzene and trichloroethene catabolism.

Rhodococcus erythropolis BD2, which is able to utilize isopropylbenzene as a sole carbon and energy source, was shown to contain a conjugative linear plasmid, pBD2. The estimated size of pBD2 is 208 to 212 kb. Linear plasmid-deficient strains had lost both the isopropylbenzene degradation and trichloroethene degradation characteristics, as well as the arsenite resistance and mercury resistance phenotypes. Reintroduction of pBD2 restored all four characteristics. Conjugational transfer of pBD2 to a plasmidless mutant of strain BD2 and other R. erythropolis strains occurred at frequencies between 3.5 x 10(-5) and 2.6 x 10(-3) transconjugants per recipient. R. erythropolis BD2 degrades isopropylbenzene via 3-isopropylcatechol and 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate. Both isopropylbenzene-oxidizing and meta-cleavage activities were shown to correspond with the presence of pBD2. Southern hybridizations with DNA encoding the toluene dioxygenase structural genes (todC1C2BA) from Pseudomonas putida F1 revealed homology to linear plasmid DNA. These results indicate that the isopropylbenzene degradation pathway encoded by linear plasmid pBD2 is initiated by an isopropylbenzene dioxygenase analogous to toluene dioxygenase.

Arsenites↗