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R Kreutzer

Publications and source records attributed to R Kreutzer.

At least 37 records · Page 2Linked to original sources
PubMed1996

Serious mushroom poisonings in California requiring hospital admission, 1990 through 1994.

Hunting and consuming wild mushrooms is an increasingly popular avocation, as well as a means of subsistence for certain groups in the United States. Consuming wild mushrooms is relatively safe for persons with the necessary expert knowledge. Each year, however, tragic deaths or illnesses occur when unsuspecting persons ingest toxic mushrooms. We conjectured that there may be vulnerable subgroups that should be targeted for public service announcements and education about mushrooms. Therefore, we reviewed California's hospital discharge database from 1990 through 1994 to ascertain demographic characteristics and outcomes of "serious" mushroom exposures requiring a hospital admission. Children younger than 5 years have a higher rate of hospital admission for "serious" poisonings. Therefore, parents should be reminded of the need for supervising children in areas with wild mushrooms. We did not find evidence that poisoning rates differed significantly between ethnic or racial groups, but this finding may be limited by a failure of these groups to seek care after becoming ill. We discuss briefly the symptoms and treatment of cases of possibly lethal ingestion of amatoxin-containing species of mushrooms.

Adolescent↗
PubMed1995

Regulation of nif gene expression in Enterobacter agglomerans: nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription.

The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3'-region of the nifM gene, the nifL and nifA genes and the 5'-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical sigma 54-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(x3) A(x3) G(x5)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH4+. Maximal promoter activity occurred at 25 degrees C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH4+ concentration in the medium exceeded 4 mM.

Amino Acid Sequence↗
PubMed1995

Site-directed mutagenesis of Thermus thermophilus EF-Tu: the substitution of threonine-62 by serine or alanine.

The invariant threonine-62, which occurs in the effector region of all GTP/GDP-binding regulatory proteins, was substituted via site-directed mutagenesis by alanine and serine in the elongation factor Tu from Thermus thermophilus. The altered proteins were overproduced in Escherichia coli, purified and characterized. The EF-Tu T62S variant had similar properties with respect to thermostability, aminoacyl-tRNA binding, GTPase activity and in vitro translation as the wild-type EF-Tu. In contrast, EF-Tu T62A is severely impaired in its ability to sustain polypeptide synthesis and has only very low intrinsic and ribosome-induced GTPase activity. The affinity of aminoacyl-tRNA to the EF-Tu T62A.GTP complex is almost 40 times lower as compared to the native EF-Tu.GTP. These observations are in agreement with the tertiary structure of EF-Tu.GTP, in which threonine-62 is interacting with the Mg2+ ion, gamma-phosphate of GTP and a water molecule, which is presumably involved in the GTP hydrolysis.

Alanine↗
PubMed1995

Site-specific mutagenesis in Enterobacter agglomerans: construction of nif B mutants and analysis of the gene's structure and function.

A novel technique was developed which may be generally well suited to the site-specific construction of mutations in Enterobacter agglomerans. The method is based on the observation that E. agglomerans can be cured of a plasmid of the incompatibility group IncQ by cultivation on citrate-containing medium. To test the applicability of this technique, we inserted a kanamycin cassette into the cloned nifB gene, transferred it into E. agglomerans, and selected for recombinants in which the wild-type nifB was replaced by the mutated gene by growing transformants on citrate medium with kanamycin. The nifB- mutants with the kanamycin cassette inserted in either orientation showed a nif- phenotype. Further, we determined the nucleotide sequence of nifB. A typical sigma 54-dependent promoter and a consensus NifA binding site were found upstream of nifB. Activation of this promoter by both heterologous and homologous NifA proteins was observed in vivo. The predicted amino acid sequence of the NifB protein showed strong similarity to the NifB sequences of other diazotrophic bacteria. The typical clustering of cysteine residues at the N-terminal end indicates its involvement in Fe-Mo cofactor biosynthesis.

Amino Acid Sequence↗
PubMed1995

Footprinting of tRNA(Phe) transcripts from Thermus thermophilus HB8 with the homologous phenylalanyl-tRNA synthetase reveals a novel mode of interaction.

The phosphates of the tRNA(Phe) transcript from Thermus thermophilus interacting with the cognate synthetase were determined by footprinting. Backbone bond protection against cleavage by iodine of the phosphorothioate-containing transcripts was found in the anticodon stem-loop, the D stem-loop and the acceptor stem and weak protection was also seen in the variable loop. Most of the protected phosphates correspond to regions around known identity elements of tRNA(Phe). Enhancement of cleavage at certain positions indicates bending of tRNAPhe upon binding to the enzyme. When applied to the three-dimensional model of tRNA(Phe) from yeast the majority of the protections occur on the D loop side of the molecule, revealing that phenylalanyl-tRNA synthetase has a rather complex and novel pattern of interaction with tRNAPhe, differing from that of other known class II aminoacyl-tRNA synthetases.

Base Composition↗
PubMed1995

Properties of isolated domains of the elongation factor Tu from Thermus thermophilus HB8.

The relative contributions of the three domains of elongation factor Tu (EF-Tu) to the factor's function and thermal stability were established by dissecting the domains apart with recombination techniques. Domain I (EF-TuI), domains I/II (EF-TuI/II) and domain III (EF-TuIII) of the EF-Tu from Thermus thermophilus HB8 comprising the amino acids 1-211, 1-312 and 317-405, respectively, were overproduced in Escherichia coli and purified. A polypeptide consisting of domain II and III (EF-TuII/III) was prepared by limited proteolysis of native EF-Tu with V8 protease from Staphylococcus aureus [Peter, M. E., Reiser, C. O. A., Schirmer, N. K., Kiefhaber, T., Ott, G., Grillenbeck, N. W. & Sprinzl, M. (1990) Nucleic Acids Res. 18, 6889-6893]. As determined by circular dichroism spectrometry, the isolated domains have the secondary structure elements found in the native EF-Tu. GTP and GDP binding as well as GTPase activity are maintained by the EF-TuI and EF-TuI/II; however, the rate of GDP dissociation from EF-TuI . GDP and EF-TuI/II . GDP complex is increased as compared to native EF-Tu . GDP, reflecting a constraint imposed by domain III on the ability to release the nucleotide from its binding pocket located in domain I. A weak interaction of Tyr-tRNATyr with the EF-TuI . GTP suggests that domain I provides a part of the structure interacting with aminoacyl-tRNA. The domain III is capable of regulating the rate of GTPase in EF-Tu, since the polypeptide consisting only of domains I/II has a 39-fold higher intrinsic GTPase compared to the native EF-Tu. No in vitro poly(U)-dependent poly(Phe) synthesis was detectable with a mixture of equimolar amounts of domains I/II and domain III, demonstrating the necessity of covalent linkage between the domains of EF-Tu for polypeptide synthesis. In contrast to native EF-Tu and EF-TuII/III, EF-TuI and, to a lesser extent the polypeptide consisting of domains I/II, are unstable at elevated temperatures. This indicates that domains II/III strongly contribute to the thermal stability of this T. thermophilus EF-Tu. Deletion of amino acid residues 181-190 from domain I of T. thermophilus EF-Tu decreases the thermostability to that of EF-Tu from E. coli, which does not have these residues. Interdomain interactions must be important for the stabilisation of the structure of domain I, since isolated T. thermophilus EF-TuI is thermolabile despite the presence of the 181-190 loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Site-directed mutagenesis of Thermus thermophilus elongation factor Tu. Replacement of His85, Asp81 and Arg300.

His85 in Thermus thermophilus elongation factor Tu (EF-Tu) was replaced by glutamine, leucine and glycine residues, leading to [H85Q]EF-Tu, [H85L] EF-Tu and [H85G]EF-Tu, respectively. Asp81 was replaced by alanine leading to [D81A]EF-Tu, and replacement of Arg300 provided [R300I]EF-Tu. Glycine in position 85 of domain I induces a protease-sensitive site in domain II and causes complete protein degradation in vivo. A similar effect was observed when Asp81 was replaced by alanine or Arg300 by isoleucine. Degradation is probably due to disturbed interactions between the domains of EF-Tu.GTP, inducing a protease-sensitive cleavage site in domain II. [H85Q]EF-Tu, which can be effectively overproduced in Escherichia coli, is slower in poly(U)-dependent poly(Phe) synthesis, has lower affinity to aminoacyl-tRNA but shows only a slightly reduced rate of intrinsic GTP hydrolysis compared to the native protein. The GTPase of this protein variant is not efficiently stimulated by aminoacyl-tRNA and ribosomes. The slow GTPase of [H85Q]EF-Tu increases the fidelity of translation as measured by leucine incorporation into poly(Phe) in in vitro poly(U)-dependent ribosomal translation. Replacement of His85 in T. thermophilus EF-Tu by leucine completely deactivates the GTPase activity but does not substantially influence the aminoacyl-tRNA binding. [H85L]EF-Tu is inactive in poly(U)-dependent poly(Phe)-synthesis. The rate of nucleotide dissociation is highest for [H85L]EF-Tu, followed by [H85Q]EF-Tu and native T. thermophilus EF-Tu. Mutation of His85, a residue which is not directly involved in the nucleotide binding, thus influences the interaction of EF-Tu domains, nucleotide binding and the efficiency and rate of GTPase activity.

Amino Acid Sequence↗
PubMed1995

Overexpression and purification of Thermus thermophilus elongation factors G, Tu, and Ts from Escherichia coli.

The translation elongation factors G (EF-G), Tu (EF-Tu), and Ts (EF-Ts) from the extreme thermophilic bacterium Thermus thermophilus were overproduced in Escherichia coli. The fus gene coding for EF-G and the tufA gene coding for EF-Tu were expressed under the control of a tac promoter, whereas EF-Ts was overproduced with the T7 RNA polymerase system. A detailed description for the purification of the three elongation factors from E. coli is presented. EF-G and EF-Tu are isolated by Q-Sepharose FF chromatography, heat treatment at 65 or 60 degrees C, respectively, and Sephacryl S200 gel permeation chromatography. For the purification of EF-Ts, a heat denaturation step is followed by DEAE-cellulose chromatography and a cation exchange EMD-SO-3 650 column. The overproduced factors show the same properties as those purified from T. thermophilus. As the crystal structures of T. thermophilus EF-Tu and EF-G have been solved recently, many questions concerning the function of particular residues or domains arise, which may be best addressed by studying the in vitro behavior and structure of altered recombinant constructs. The methods presented here should facilitate such studies.

Bacteriophage T7↗
PubMed1994

Expression cloning of the nox, mdh and ldh genes from Thermus species encoding NADH oxidase, malate dehydrogenase and lactate dehydrogenase.

The Thermus thermophilus HB8 mdh and ldh genes and the T. aquaticus EP00276 nox and mdh genes encoding the biotechnologically important enzymes NADH oxidase (EC 1.6.99.3), malate dehydrogenase (EC 1.1.1.37) and lactate dehydrogenase (EC 1.1.1.27) were cloned on the basis of known sequences from related species using the polymerase chain reaction. The nox and mdh genes were directly placed under the control of regulatory expression elements from Escherichia coli. When the 5'-portions of the re-cloned nox gene and the mdh gene of T. thermophilus HB8 were simultaneously altered, enzyme yields of 18-42% of the total soluble cellular protein were obtained as compared to 2-6% obtained from the unchanged genes. The high overproduction level upon the alterations can be explained by the occurrence of additional potential base pairs between nucleotides in the mRNA downstream of the start codon ('downstream box') and the 16S rRNA. An 'universal translation initiation sequence' providing such strong interactions may be of general use for high overproduction levels.

Base Sequence↗
PubMed1993

Sequence, overproduction and crystallization of aspartyl-tRNA synthetase from Thermus thermophilus. Implications for the structure of prokaryotic aspartyl-tRNA synthetases.

The genes of aspartyl-tRNA synthetase (AspRS) from two Thermus thermophilus strain VK-1 and HB8, have been cloned and sequenced. Their nucleotidic sequences code for the same protein which displays the three characteristic motifs of class II aminoacyl-tRNA synthetases. This enzyme shows 50% identity with Escherichia coli AspRS, over the totality of the chain (580 amino acids). A comparison with the eukaryotic yeast cytoplasmic AspRS indicates the presence in the prokaryotic AspRS of an extra domain between motifs 2 and 3 much larger than in the eukaryotic ones. When its gene is under the control of the tac promoter of the expression vector pKK223-3, the protein is efficiently overexpressed as a thermostable protein in E. coli. It can be further purified to homogeneity using a heat treatment followed by a single anion exchange chromatography. Single crystals of the pure protein, diffracting at least to 2.2 A resolution (space group P2(1)2(1)2(1), a = 61.4 A, b = 156.1 A, c = 177.3 A) are routinely obtained. The same crystals have previously been described as crystals of threonyl-tRNA synthetase [1].

Amino Acid Sequence↗
PubMed1993

Participation of the overproduced elongation factor Tu from Thermus thermophilus in protein biosynthesis of Escherichia coli.

The influence of the overproduced elongation factor Tu (EF-Tu) from Thermus thermophilus on the protein biosynthesis in Escherichia coli was investigated both in vivo and in vitro. A kirromycin-resistant E. coli strain became sensitive to this antibiotic upon the expression of the tuf A-gene of T. thermophilus present on a plasmid. In in vitro translation with components of the kirromycin-resistant E. coli strain the poly(Phe) synthesis stopped when minute amounts of the EF-Tu from T. thermophilus were added. Both results indicate the sensitivity of the T. thermophilus EF-Tu to kirromycin and its participation in the polypeptide synthesis of E. coli.

Bacterial Proteins↗
PubMed1992

Structure of the phenylalanyl-tRNA synthetase genes from Thermus thermophilus HB8 and their expression in Escherichia coli.

A 4459 bp long BamHI restriction fragment containing the two genes for the Thermus thermophilus HB8 phenylalanyl-tRNA synthetase was cloned in Escherichia coli and its nucleotide sequence was determined. The genes pheS and pheT encode the alpha- and beta-subunits with a molecular weight of 39 and 87 kD, respectively. Three conserved sequence motifs typical for class II tRNA synthetases occur in the alpha-subunit. Secondary structure predictions indicate that an arm composed of two anti-parallel alpha-helices similar to that reported for the E.coli seryl-tRNA synthetase may be present in its N-terminal portion. In the beta-subunit clusters of hydrophilic amino acids and a leucine zipper motif were identified, and several pronounced alpha-helical regions were predicted. The particular arginine and lysine residues in the N-terminal portion of the beta-subunit, which were found to participate in tRNA binding in the yeast and E.coli PheRSs, have their counterparts in the T.thermophilus protein. The 5'-portion of an open reading frame downstream of pheT was found and codes for a yet unidentified, extremely hydrophobic peptide. The pheST genes are presumably cotranscribed and translationally coupled. A novel type of a putative transcriptional terminator in Thermus species was identified immediately downstream of pheT and other Thermus genes. The genes pheS and pheST were expressed in E.coli.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and nucleotide sequence of the gene encoding a H2O2-forming NADH oxidase from the extreme thermophilic Thermus thermophilus HB8 and its expression in Escherichia coli.

The sequence of the 32 N-terminal amino acids of the NADH oxidase from the extreme thermophile, Thermus thermophilus HB8, was used to synthesize oligonucleotides to probe for the respective gene in a genomic library of T. thermophilus HB8. The gene encoding the NADH oxidase, designated nox, was cloned, its nucleotide sequence was determined and found to be colinear with the N-terminal sequence of the enzyme. The molecular mass of 26835 Da, as deduced from the nox gene, agrees with that of the purified NADH oxidase from T. thermophilus HB8 (25,000 Da), as estimated by polyacrylamide gel electrophoresis under denaturing conditions. The nox gene was overexpressed in Escherichia coli and a protocol for the rapid purification of the enzyme was developed. The E. coli-borne T. thermophilus HB8 NADH oxidase has properties identical to those of the authentic T. thermophilus HB8 enzyme and possesses a high thermal stability.

Amino Acid Sequence↗
PubMed1991

Leishmania donovani chagasi: new clinical variant of cutaneous leishmaniasis in Honduras.

During surveillance for endemic visceral leishmaniasis on an island off the Pacific coast of Honduras, an unusual form of cutaneous leishmaniasis was encountered. By clinical and laboratory criteria, 17 cases were identified over 5 months; children aged 4 to 15 years were primarily affected. Lesions were generally few in number, small, always papular, and non-ulcerative, even when present for several years. Patients with skin lesions seemed otherwise healthy and were well nourished. Montenegro skin tests with Leishmania mexicana and L major antigens were positive in 10 of 17 patients tested, and lesions from 9 patients were positive by culture. Since the summer of 1988, cases of atypical cutaneous leishmaniasis continue to occur on the island (8) as well as on the mainland of southern Honduras (23). A total of 9 parasite isolates from skin lesions, 4 from bone marrow of patients with kala-azar, and 2 from sandflies were identified as L donovani chagasi and were indistinguishable from one another by isoenzyme analysis.

Adolescent↗
PubMed1991

Identification of a promoter dependent on NifA and sigma 54 upstream of nifH in Azospirillum lipoferum.

Southern hybridization experiments strongly indicate that the regulatory region of the Azospirillum lipoferum nifH gene is located on a cloned 1.1 kb BamHI-XhoI restriction fragment. By cloning this fragment into a promoter-probe plasmid in Escherichia coli, a promoter was identified oriented towards the nifH gene. Using a set of several bacterial strains and plasmids, both NifA and the alternative sigma factor, sigma 54, from Klebsiella pneumoniae were shown to be required for the induction of the assumed nifH promoter in this particular heterologous system. However, NtrC from K. pneumoniae did not stimulate this promoter. No other promoter activity was detected in the direction opposite to the identified promoter, indicating that the transcription of the adjacent nifJ gene cannot be initiated from the 1.1 kb BamHI-XhoI fragment. Thus, the genes nifH and nifJ in A. lipoferum cannot be oriented divergently, in contrast to the situation in several other nitrogen-fixing bacteria.

Azospirillum brasilense↗
PubMed1991

Cotranscription of the electron transport protein genes nifJ and nifF in Enterobacter agglomerans 333.

A nucleotide sequence showing extensive homology to the nifF gene, which codes for a flavodoxin involved in nitrogen fixation in Klebsiella pneumoniae, was localized on the plasmid pEA3 of Enterobacter agglomerans and determined. The analysis of transcriptional fusions, as well as transcript protection assays, indicated a novel nif gene organization, that is, the cotranscription of nifJ and nifF.

Amino Acid Sequence↗