PubMed Health⌕ Search

Biomedical subjects

T Leisinger

Publications and source records attributed to T Leisinger.

At least 91 records · Page 5Linked to original sources
PubMed1988

Identification of a transcript and its promoter region on the archaebacterial plasmid pME2001.

The cryptic multicopy plasmid pME2001 of Methanobacterium thermoautotrophicum Marburg encodes a 611-base-pair transcript containing several consecutive, short open reading frames. Scrutiny of the 5'-flanking region did not reveal homology to putative archaebacterial consensus promoter sequences. However, 28 base pairs upstream of the transcription start point, there was a sequence with strong homology to a sequence preceding the purE gene of M. thermoautotrophicum.

Base Sequence↗
PubMed1988

Dichloromethane dehalogenase with improved catalytic activity isolated from a fast-growing dichloromethane-utilizing bacterium.

A methylotrophic bacterium, denoted strain DM11, was isolated from groundwater and shown to utilize dichloromethane or dibromomethane as the sole carbon and energy source. The new isolate grew at the high rate of 0.22 h-1 compared with 11 previously characterized dichloromethane-utilizing bacteria (micromax, 0.08 h-1). The dichloromethane dehalogenase from strain DM11 (group B enzyme) was purified by anion-exchange chromatography. It was shown to be substantially different from the set of dichloromethane dehalogenases from the 11 slow-growing strains (group A enzymes) that had previously been demonstrated to be identical. The Vmax for the group B enzyme was 97 mkat/kg of protein, some 5.6-fold higher than that of the group A enzymes. The group A dehalogenases showed hyperbolic saturation with the cosubstrate glutathione, whereas the group B enzyme showed positive cooperativity in glutathione binding. Only 1 of 15 amino acids occupied common positions at the N termini, and amino acid contents were substantially different in group A and group B dehalogenases. Immunological assays demonstrated weak cross-reactivity between the two enzymes. Despite the observed structural and kinetic differences, there is potentially evolutionary relatedness between group A and group B enzymes, as indicated by (i) hybridization of DM11 DNA with a gene probe of the group A enzyme, (ii) a common requirement for glutathione in catalysis, and (iii) similar subunit molecular weights of about 34,000.

Amino Acid Sequence↗
PubMed1988

Anabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa: nucleotide sequence and transcriptional control of the argF structural gene.

In Pseudomonas aeruginosa PAO the anabolic ornithine carbamoyltransferase (OTCase, EC 2.1.3.3) is the product of the argF gene and the only arginine biosynthetic enzyme whose synthesis is repressible by arginine. We have determined the complete nucleotide sequence of the argF gene including its promoter-control region. The deduced amino acid sequence of the anabolic OTCase consists of 305 residues (Mr 33,924), and this was confirmed by the N-terminal amino acid sequence, the total amino acid composition, and the subunit Mr of the purified enzyme. The native anabolic OTCase (Mr 110,000 to 125,000) was found to be a trimer by cross-linking experiments. P. aeruginosa also has a catabolic OTCase (the arcB gene product), which catalyzes the reverse reaction of the anabolic conversion. At the nucleotide sequence level, the P. aeruginosa argF gene had 52.4% identity with the arcB gene. The Escherichia coli argF and argI genes, which code for anabolic OTCase isoenzymes, had 47.3 and 44.9% identity, respectively, with the P. aeruginosa argF sequence. This suggests that these four genes have evolved from a common ancestral gene. The arcB gene appears to be more closely related to the E. coli argF gene than to the P. aeruginosa argF gene. Two transcripts (mRNA-1, mRNA-2) of the P. aeruginosa argF gene were identified by S1 mapping. The transcription initiation site for mRNA-1 was preceded by sequences having partial homology with the E. coli -35 and -10 consensus promoter sequences. No sequence similar to consensus promoters of enteric bacteria was found upstream of the 5' end of mRNA-2. E. coli carrying a P. aeruginosa argF+ recombinant plasmid produced mRNA-1 with low efficiency but no (or very little) mRNA-2. Arginine repressed argF transcription in P. aeruginosa. In the argF promoter region no sequence homologous to the "arg box" (arginine operator module) of E. coli was found. The mechanism of arginine repression in P. aeruginosa thus appears to be different from that in E. coli.

Amino Acid Sequence↗
PubMed1987

Microbial desulfonation of substituted naphthalenesulfonic acids and benzenesulfonic acids.

Sulfur-limited batch enrichment cultures containing one of nine multisubstituted naphthalenesulfonates and an inoculum from sewage yielded several taxa of bacteria which could quantitatively utilize 19 sulfonated aromatic compounds as the sole sulfur source for growth. Growth yields were about 4 kg of protein per mol of sulfur. Specific degradation rates were about 4 to 14 mu kat/kg of protein. A Pseudomonas sp., an Arthrobacter sp., and an unidentified bacterium were examined. Each desulfonated at least 16 aromatic compounds, none of which served as a carbon source. Pseudomonas sp. strain S-313 converted 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, 5-amino-1-naphthalenesulfonic acid, benzenesulfonic acid, and 3-aminobenzenesulfonic acid to 1-naphthol, 2-naphthol, 5-amino-1-naphthol, phenol, and 3-aminophenol, respectively. Experiments with 18O2 showed that the hydroxyl group was derived from molecular oxygen.

Arthrobacter↗
PubMed1987

Characterization of 1-chlorohexane halidohydrolase, a dehalogenase of wide substrate range from an Arthrobacter sp.

1-Chlorohexane halidohydrolase from Arthrobacter sp. strain HA1 was purified to homogeneity by fractional precipitation, ion-exchange chromatography, gel filtration, and high-performance liquid chromatography gel filtration. The enzyme was a monomer with a molecular weight of about 37,000; its amino acid composition and N-terminal sequence were determined. The enzyme had a broad optimum around pH 9.5, a temperature optimum near 50 degrees C, an activation energy of 40 kJ/mol, and a molecular activity of 0.9 kat/mol. The substrate range of the enzyme included at least 50 halogenated compounds. 1-Chloroalkanes (C3 to C10), 1-bromoalkanes (C1 to C9), and 1-iodoalkanes (C1 to C7), but no 1-fluoroalkane, were substrates. Subterminally substituted, branched-chain, and nonsaturated haloalkanes were dehalogenated. Some halogenated aromatic substrates, e.g., bromobenzene and benzyl bromide, were hydrolyzed. Several alpha,omega-dihaloalkanes were subject to double dehalogenation. Thus, 1,2-dibromoethane was hydrolyzed first to 2-bromoethanol and then to 1,2-dihydroxyethane. Crude extracts of strain HA1 were found to contain a debrominase that cleaved bromoalkanes with long alkyl chains.

Amino Acid Sequence↗
PubMed1987

Purification and use of Methanobacterium wolfei pseudomurein endopeptidase for lysis of Methanobacterium thermoautotrophicum.

The pseudomurein-degrading enzyme from autolysates of Methanobacterium wolfei was purified approximately 500-fold to electrophoretic homogeneity by ion-exchange chromatography under anaerobic conditions. Analysis of the soluble cell wall fragments produced by the pure enzyme from a cell wall preparation of M. thermoautotrophicum indicated that it is a peptidase hydrolyzing the epsilon-Ala-Lys bond of pseudomurein. A partially purified preparation of pseudomurein endopeptidase was free of nuclease activity and thus proved useful for the preparation in high yields of undegraded chromosomal and plasmid DNA from M. thermoautotrophicum. The partially purified enzyme was also used for the preparation of protoplasts, which were stabilized by 0.8 M sucrose. Under growth conditions the protoplasts produced methane and increased up to 100-fold in size, but failed to regenerate a cell wall.

Endopeptidases↗
PubMed1986

Expression of biosynthetic genes from Pseudomonas aeruginosa and Escherichia coli in the heterologous host.

We examine the expression of constitutive or repressible, monocistronic genes from Pseudomonas aeruginosa and Escherichia coli after their transfer to the heterologous host. To this end, chromosomal DNA from P. aeruginosa was cloned into the mobilizable broad-host-range vector pKT240; recombinant plasmids carrying the argA, argF, or proC genes were identified by complementation of the corresponding auxotrophic mutations. The isofunctional E. coli genes and the E. coli proB gene were subcloned into pKT240 from existing recombinant plasmids. The enzyme expression specified by the Pseudomonas genes in E. coli, calculated per gene copy, ranged from 0.3%-5% of the levels observed in Pseudomonas. Fusion of the P. aeruginosa proC gene to the E. coli consensus tac promoter resulted in very high proC enzyme production in E. coli, indicating that, at least in this case, the expression barrier is essentially at the level of transcriptional initiation. The E. coli argA and argF enzymes, which are controlled by repression in their native host, were synthesized constitutively in P. aeruginosa at 5% of the levels measured in E. coli under derepressed conditions. The constitutive E. coli proB and proC genes were expressed at high levels (ca. 50%) in the heterologous host. These results support the idea that P. aeruginosa may be a more permissive host than E. coli for the heterologous expression of genes from gram-negative bacteria.

Chromosomes, Bacterial↗
PubMed1986

N-Succinylated intermediates in an arginine catabolic pathway of Pseudomonas aeruginosa.

Arginine-nonutilizing (aru) mutants of Pseudomonas aeruginosa strain PAO converted L-arginine to N(2)-succinylarginine or N-succinylglutamate, which were identified by high-voltage electrophoresis and HPLC. Addition of aminooxyacetate, an inhibitor of pyridoxal phosphate-dependent enzymes, to resting cells of the wild-type PAO1 in arginine medium led to the accumulation of N(2)-succinylornithine. Enzyme assays with crude P. aeruginosa extracts established the following pathway: L-arginine + succinyl-CoA --> N(2)-succinylarginine --> N(2)-succinylornithine --> N_succinylglutamate 5-semialdehyde --> N-succinylglutamate --> succinate + glutamate. Succinyl-CoA may be regenerated from glutamate via 2-ketoglutarate. L-Arginine induced the enzymes of the pathway, and succinate caused catabolite repression. Purified N(2)-acetylornithine 5-aminotransferase (N(2)-acetyl-L-ornithine: 2-oxoglutarate aminotransferase, EC 2.6.1.11), an arginine biosynthetic enzyme, efficiently transaminated N(2)-succinylornithine; this explains the enzyme's dual role in arginine biosynthesis and catabolism. The succinylarginine pathway enables P. aeruginosa to utilize arginine efficiently as a carbon source under aerobic conditions, whereas the other three arginine catabolic pathways previously established in P. aeruginosa fulfill different functions.

Journal Article↗
PubMed1985

Cloning of DNA sequences from Methanococcus vannielii capable of autonomous replication in yeast.

Total DNA of the archaebacterium Methanococcus vannielii was digested with BamHI or BamHI/HindIII, cloned with plasmid Yip5 and analyzed for sequences capable of autonomous replication (ARSs) in the eukaryote Saccharomyces cerevisiae. Two recombinant plasmids were isolated which contained 3.3 kb and 8 kb fragments of methanogen derived DNA with ARS activity. They exhibited low transformation efficiencies for yeast and promoted slow growth of yeast transformants.

Cloning, Molecular↗
PubMed1985

Altered control of glutamate dehydrogenases in ornithine utilization mutants of Pseudomonas aeruginosa.

Two classes of ornithine-nonutilizing (oru) mutants of Pseudomonas aeruginosa PAO were investigated. Strains carrying the oru-310 mutation were entirely unable to grow on L-ornithine as the only carbon and nitrogen source and were affected in the assimilation of a variety of nitrogen sources (e.g., amino acids, nitrate). The oru-310 mutation caused changes in the regulation of the catabolic NAD-dependent glutamate dehydrogenase; this enzyme was no longer inducible by glutamate but instead could be induced by ammonia. The oru-310 locus was cotransducible with car-9 and tolA in the 10 min region of the chromosome. An oru-314 mutant was severely handicapped in ornithine medium but could grow when a good carbon source was added; the mutant also showed pleiotropic growth effects related to nitrogen metabolism. The oru-314 mutation affected the regulation of the anabolic NADP-dependent glutamate dehydrogenase, which was no longer repressed by glutamate but showed normal derepression in the presence of ammonia. The oru-314 locus was mapped by transduction near met-9011 at 55 min. Both oru mutants could grow on L-glutamate, L-proline, or L-ornithine amended with 2-oxoglutarate, albeit slowly. We speculate that insufficient 2-oxoglutarate concentrations might account, at least in part, for the Oru- phenotype of the mutants.

Chromosome Mapping↗
PubMed1985

Dichloromethane dehalogenase of Hyphomicrobium sp. strain DM2.

Dichloromethane dehalogenase, a highly inducible glutathione-dependent enzyme catalyzing the conversion of dichloromethane into formaldehyde and inorganic chloride, was purified fivefold with 60% yield from Hyphomicrobium sp. strain DM2. The electrophoretically homogeneous purified enzyme exhibited a specific activity of 17.3 mkat/kg of protein. Its pH optimum was 8.5. The enzyme was stable at -20 degrees C for at least 6 months. A subunit molecular weight of 33,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration of native dichloromethane dehalogenase yielded a molecular weight of 195,000. Subunit cross-linking with dimethyl suberimidate confirmed the hexameric tertiary structure of the enzyme. Dichloromethane dehalogenase was highly specific for dihalomethanes. Its apparent Km values were 30 microM for CH2Cl2, 15 microM for CH2BrCl, 13 microM for CH2Br2, 5 microM for CH2I2, and 320 microM for glutathione. Several chlorinated aliphatic compounds inhibited the dichloromethane dehalogenase activity of the pure enzyme. The Ki values of the competitive inhibitors 1,2-dichloroethane and 1-chloropropane were 3 and 56 microM, respectively.

Bacteria↗
PubMed1984

Revised locations of the hisI and pru (proline utilization) genes on the Pseudomonas aeruginosa chromosome map.

The location of genes in the vicinity of the major FP2 origin on the chromosome of Pseudomonas aeruginosa PAO has been revised. The markers hisI (a transduction group of histidine biosynthetic genes) and pru (a gene cluster encoding proline utilization functions) were located in the 90 to 95/0 min chromosome region by a series of plate matings mediated by R68.45. Three-factor-crosses using this plasmid established the following marker order: pur-67 pru hisI/cys-59 proB ilvB/C. Genetic evidence is presented to confirm the previous observations that FP2 can mobilize the chromosome from at least two origins near proB and in both directions. Thus, when markers in this chromosome region are analyzed by FP2 crosses only, the mapping data may be difficult to interpret. This complication can be overcome by the use of R68.45 and Tfr (transposon-facilitated recombination) or Hfr donors.

Chromosome Mapping↗
PubMed1984

Comparison of two bacterial azoreductases acquired during adaptation to growth on azo dyes.

Selection for utilization of carboxy-Orange I [1-(4'-carboxyphenylazo)-4-naphthol] in the chemostat yielded Pseudomonas strain K24 which was unable to grow on carboxy-Orange II [1-(4'-carboxyphenylazo)-2-naphthol] while selection for growth on carboxy-Orange II had previously led to strain KF46 which did not utilize carboxy-Orange I. Orange I azoreductase of strain K24, the key enzyme of dye degradation, was purified 80-fold with 17% yield to electrophoretic homogeneity and compared to the previously purified Orange II azoreductase of strain KF46. Common properties of the two enzymes were their monomeric structure, their specificity for NADPH and NADH as cosubstrates, the range of their Km values for substrates and cosubstrates as well as their reactivity towards a series of substrate analogs. They differed from each other with respect to molecular weight (21,000 and 30,000) and in the absolute requirement of Orange I azoreductase for a hydroxy group in the 4'position of the naphthol ring of the substrate molecule as compared to the requirement for substrates with a 2-naphthol moiety by Orange II azoreductase. The pure enzymes did not exhibit immunological cross-reaction with each other. Crude extracts of strains K24 and KF46 and of azoreductase-negative strains isolated at different stages of the adaptation experiments, however, contained material which cross-reacted (CRM) with both anti Orange I azoreductase serum and anti Orange II azoreductase serum. The CRM may represent a common precursor protein of the azoreductases in strains K24 and KF46.

Adaptation, Physiological↗
PubMed1984

Genetic and molecular characterization of the Pseudomonas plasmid pVS1.

A restriction map of the 30-kb nonconjugative Pseudomonas plasmid pVS1 was constructed. Derivatives of pVS1 obtained in vitro by successive deletions were used to localize on the physical map the determinant for resistance to mercuric ions (carried by transposon Tn501), the gene(s) encoding sulfonamide resistance, a 1.6-kb region affecting plasmid stability and establishment in P. fluorescens ATCC 13525, and a segment required for mobilization of pVS1 by plasmid RP1. The sulfonamide resistance determinant of pVS1 appeared to be closely related to that of transposon Tn21. A mini-pVS1 replicon, pME259, consisting of an essential 1.55-kb segment (designated rep and thought to carry the origin of replication) and a mercury resistance determinant was able to replicate P. aeruginosa PAO but selective pressure was needed for plasmid maintenance. The copy number of pVS1 derivatives was estimated to be 6-8 per chromosome equivalent. Plasmids possessing the essential rep segment plus the adjacent stability region could be established in strains of P. aeruginosa, P. putida, P. fluorescens, P. acidovorans, P. cepacia, P. mendocina, P. stutzeri, P. syringae, Agrobacterium tumefaciens, and Rhizobium leguminosarum.

DNA Replication↗
PubMed1983

Bacterial growth on 1,2-dichloroethane.

1,2-Dichloroethane (5 mM) served as the only carbon and energy source for bacterium DE2, a gram-negative, oxidase-positive, motile rod. The specific growth rate mu of strain DE2 on 1,2-dichloroethane was 0.08 h-1. A NAD-dependent 2-chloroacetaldehyde dehydrogenase activity and a 2-chloroacetate halidohydrolase activity were detected in extracts of cells grown on 1,2-dichloroethane.

Aldehyde Oxidoreductases↗
PubMed1983

Proline biosynthesis in Escherichia coli. Kinetic and mechanistic properties of glutamate semialdehyde dehydrogenase.

The kinetics of the NADP+- and phosphate-dependent oxidation of glutamic acid 5-semialdehyde are consistent with a rapid-equilibrium random order mechanism. The Km for DL-pyrroline-5-carboxylic acid is 2.5 mM, for NADP+ is 0.05 mM and for phosphate is 0.35 mM. The Vmax is approx. 8.0 units per mg protein. The reaction is highly specific for the DL-pyrroline-5-carboxylic acid and NADP+, but a number of divalent anions can substitute for phosphate. NADPH is competitive with respect to all three substrates and an analog of gamma-glutamyl phosphate, 3-(phosphonoacetylamido)-L-alanine, is competitive with respect to DL-pyrroline-5-carboxylic acid and non-competitive with respect to NADP+ and phosphate, suggesting dead-end complex formation.

Aldehyde Oxidoreductases↗