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R Krämer

Publications and source records attributed to R Krämer.

At least 19 recordsLinked to original sources
PubMed2004

Projection structure and oligomeric state of the osmoregulated sodium/glycine betaine symporter BetP of Corynebacterium glutamicum.

The high-affinity glycine betaine uptake system BetP, an osmosensing and osmoregulated sodium-coupled symporter from Corynebacterium glutamicum, was overexpressed in Escherichia coli with an N-terminal StrepII-tag, solubilized in beta-dodecylmaltoside and purified by streptactin affinity chromatography. Analytical ultracentrifugation indicated that BetP forms trimers in detergent solution. Detergent-solubilized BetP can be reconstituted into proteoliposomes without loss of function, suggesting that BetP is a trimer in the bacterial membrane. Reconstitution with E.coli polar lipids produced 2D crystals with unit cell parameters of 182A x 154A, gamma=90 degrees exhibiting p22(1)2(1) symmetry. Electron cryo-microscopy yielded a projection map at 7.5A. The unit cell contains four non-crystallographic trimers of BetP. Within each monomer, ten to 12 density peaks characteristic of transmembrane alpha-helices surround low-density regions that define potential transport pathways. Small but significant differences between the three monomers indicate that the trimer does not have exact 3-fold symmetry. The observed differences may be due to crystal packing, or they may reflect different functional states of the transporter, related to osmosensing and osmoregulation. The projection map of BetP shows no clear resemblance to other secondary transporters of known structure.

Affinity Labels↗
PubMed2002

GltS, the sodium-coupled L-glutamate uptake system of Corynebacterium glutamicum: identification of the corresponding gene and impact on L-glutamate production.

A screening procedure was established to identify Corynebacterium glutamicum transposon mutants with an altered L-glutamate excretion behaviour. By this microtiter plate-based approach seven non- or less excreting C. glutamicum strains and two hyper-excreters were found. The subsequently carried out molecular analysis of a hyper-producing clone led to the identification of the gltS gene, which codes for the sodium-coupled secondary L-glutamate uptake system in C. glutamicum. Characterization of a gltS deletion strain revealed that this transporter has a weak but significant impact on L-glutamate production induced by biotin limitation in the wild type. Obviously, GltS leads to the re-uptake of excreted L-glutamate causing a futile cycle. In accord with this hypothesis, the overexpression of gltS decreased L-glutamate production.

Amino Acid Transport Systems, Acidic↗
PubMed2002

Influence of threonine exporters on threonine production in Escherichia coli.

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [(14)C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.

Amino Acid Transport Systems, Neutral↗
PubMed2002

A new technique for the surgical management of deformities in the growing spine.

The Luque procedure was developed to correct the deformity without the need of bracing and maintaining that correction with growth. However many authors are disappointed by their results and the complications which appear in the management of infantile scoliosis with Luque trolley alone. Besides failed implants, pseudarthrosis, modest spinal growth and protuberant rods and wires, the major problem of the Luque systems is the high incidence of loss of correction by postoperative rotation. Therefore a new application technique is recommended. A standard posterior extraperiostal approach is chosen. Sublaminar titanium cables are passed at each level except the caudal lamina. Then the rods are precontoured in shape of the planed curve correction. We use a low profile titanium instrumentation with 5.0 mm diameter rods and 4.2 mm pedicle screws. In contrast to the conventional use of two antiparallel "L"-rods we recommend the use of one reversed "U"-rod securing the laminae with sublaminar titanium cables of the upper end vertebrae. For fixation of the lower spine a dual-opening pedicle screw system is used. Using a holding forceps the distal rods are introduced and fixed into the side opening of the screws then secured by sublaminar wires. In addition both single rods are stabilized by a low profile cross link bar. This technique allows to correct pelvic obliquity and a stable anchorage of two screws reduces risk of postoperatively rotation or caudal rod slippage due to gravity forces.

Adolescent↗
PubMed2002

The effect of limited interlaminar decompression versus complete laminectomy on intrathecal volume in degenerative lumbar spinal stenosis.

INTRODUCTION: There is a controversial discussion about the adequate surgical procedure for degenerative lumbar spinal stenosis. Due to the observation that the degenerative lumbar spinal stenosis takes place predominantly at the interlaminar region on the level of the disc involving facets and bulging of the ligamentum flavum, resection of the whole lamina might not be necessary. A biomechanical study was designed to assess the effect of different decompression techniques using cadaver lumbar spine models. METHODS: Twelve cadaver spines with CT verified degenerative lumbar spinal stenosis were dissected in order to measure the volume of the dural sac at different flexion and extension angles. Each segment (L3/4, L4/5) was decompressed first by limited interlaminar decompression and second by complete laminectomy. Intrathecal volume measurements were taken initially, after limited interlaminar decompression and after complete laminectomy. RESULTS: Before surgical procedure, the cadaver spines showed an increase of the intrathecal volume in flexion and decrease in extension. After limited interlaminar decompression, there was a significant reduction of volume loss in extension. There was no significant additional reduction of volume loss in extension after complete laminectomy in comparison to limited interlaminar decompression. CONCLUSION: The results allow to conclude that limited interlaminar decompression is efficient for decompression in degenerative lumbar spinal stenosis.

Aged↗
PubMed2002

Experience with integrated water quality management in the Wahnbach Watershed.

This paper describes how integrated water quality management has been developed for Wahnbach reservoir and the experience which has been gained with this multi-barrier system. Different measures in the catchment area, where co-operation with farmers was most important, are combined with the operation of a phophorus elimination plant, in which the main tributary is treated, and with aeration and biomanipulation in the reservoir. All measures together have led to the present oligotrophic state and the excellent hygienic condition of the reservoir.

Conservation of Natural Resources↗
PubMed2001

Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applications.

Transport processes play a pivotal role in cellular metabolism, e.g. for the uptake of nutrients or the excretion of metabolic waste products. Moreover, they are also important in biotechnological processes such as the production of various amino acids by the use of microorganisms. The focus of this review is on bacterial amino acid transport systems, in particular those of Corynebacterium glutamicum and Escherichia coli, with respect to their function and biotechnological significance.

Amino Acid Transport Systems↗
PubMed2001

[Occupational lead poisoning due to deficient protective measures at the work place].

UNLABELLED: Occupational lead poisoning due to deficient protective measures at the work place. HISTORY AND ADMISSION FINDINGS: A 27-year-old man who was working in the mixing plant of a company manufacturing synthetic materials, was admitted with colicky upper abdominal pain, loss of appetite and postprandial nausea for 3 weeks. On examination he had pain without guarding over the abdomen. INVESTIGATIONS: He had a normochronic anaemia (haemoglobin 12.5. g/dl). Ultrasound of the abdomen revealed splenomegaly. Gastroscopy excluded an ulcer. Deep duodenal biopsy showed Giardia intestinalis histologically. DIAGNOSIS, TREATMENT AND COURSE: As part of the differential diagnosis of abdominal colic and anaemia, lead poisoning was found, with the lead level markedly elevated to 1776 microgram/l. Penicillamine treatment rapidly reduced the blood lead level, but it had not yet become normal after 4 months. Exposure to lead at his work place was discovered. Subsequent tests there by the federal agency for occupational protection revealed massive deficiencies in protective measures. CONCLUSION: In present conditions abdominal colic must be viewed as a cardinal symptom of lead poisoning. Blood levels must be obtained as part of the differential diagnosis of abdominal pain of uncertain aetiology. If the diagnosis has been established and an occupational risk is possible, the appropriate occupational organizations and, if necessary, federal agency must be informed in order to exclude occupational exposure to lead. In Germany there still exist marked deficiencies in the protection of workers occupationally exposed to lead. Notification is essential to protect others similarly at risk.

Adult↗
PubMed2001

The osmoreactive betaine carrier BetP from Corynebacterium glutamicum is a sensor for cytoplasmic K+.

The isolated glycine betaine uptake carrier BetP from Corynebacterium glutamicum was reconstituted in Escherichia coli phospholipid liposomes and its response to osmotic stress studied. The transport activity of BetP, which was previously shown to comprise both osmosensory and osmoregulatory functions, was used to identify the nature of the physicochemical stimulus related to hyperosmotic stress. Putative factors modulating transport activity in response to osmotic stress were dissected. These include type, osmolality and concentration of solutes in the internal and/or external compartment (cationic, anionic, zwitterionic, neutral), as well as membrane strain as a response to increased osmolality. Osmoresponsive activation of BetP was independent of any external factor and of physical alterations of the membrane, but was triggered by a change in the internal K+ concentration. Activation did not depend on the type of anion present and was K+ (or Cs+ and Rb+) specific, as choline and NH(4)+ did not trigger BetP activity. The half-maximal activation of BetP in E.coli phospholipid liposomes was correlated to an internal concentration of 221 +/- 23 mM K+.

Bacterial Proteins↗
PubMed2001

The putative monocarboxylate permeases of the yeast Saccharomyces cerevisiae do not transport monocarboxylic acids across the plasma membrane.

We have characterized the monocarboxylate permease family of Saccharomyces cerevisiae comprising five proteins. We could not find any evidence that the monocarboxylate transporter-homologous (Mch) proteins of S. cerevisiae are involved in the uptake or secretion of monocarboxylates such as lactate, pyruvate or acetate across the plasma membrane. A yeast mutant strain deleted for all five MCH genes exhibited no growth defects on monocarboxylic acids as the sole carbon and energy sources. Moreover, the uptake and secretion rates of monocarboxylic acids were indistinguishable from the wild-type strain. Additional deletion of the JEN1 lactate transporter gene completely blocked uptake of lactate and pyruvate. However, uptake of acetate was not even affected after the additional deletion of the gene YHL008c, which had been proposed to code for an acetate transporter. The mch1-5 mutant strain showed strongly reduced biomass yields in aerobic glucose-limited chemostat cultures, pointing to the involvement of Mch transporters in mitochondrial metabolism. Indeed, intracellular localization studies indicated that at least some of the Mch proteins reside in intracellular membranes. However, pyruvate uptake into isolated mitochondria was not affected in the mch1-5 mutant strain. It is concluded that the yeast monocarboxylate transporter-homologous proteins perform other functions than do their mammalian counterparts.

Biomass↗
PubMed2001

Glutamine synthetases of Corynebacterium glutamicum: transcriptional control and regulation of activity.

Regulation of glnA expression and glutamine synthetase I activity was analyzed in Corynebacterium glutamicum. Transcription is regulated by the global repressor protein AmtR, essential for derepression of glnA transcription are GlnK and uridylyltransferase, key proteins of the C. glutamicum nitrogen regulatory system. Glutamine synthetase I activity is controlled by adenylylation/deadenylylation via adenylyltransferase. The gene encoding this bifunctional enzyme, glnE, was isolated and its function was characterized by deletion analysis. Upstream of glnE, a second gene encoding a GSI-type protein in C. glutamicum was isolated. This gene, designated glnA2, forms an operon with glnE, its transcription is not regulated and neither its deletion or overexpression showed any effect. Therefore, the physiological role of glnA2 remains unclear.

Bacterial Proteins↗
PubMed2001

An allosteric synthetic catalyst: metal ions tune the activity of an artificial phosphodiesterase.

A trinuclear metal complex of general formula (L-H)M3(Mf)2 represents the first allosteric low molecular weight catalyst. L is a polyaza ligand having a tetradentate and two bidentate metal binding sites, Ms is a "structural" (allosteric) metal, and Mf are functional (catalytic) metals which interact with a substrate. In mononuclear [(L-H)Ms]+ complexes [(L-H)Cu(MeOH)]ClO4 (1a). [(L-H)Cu]NO3 x 2H2O (1b), [(L-H)Ni]ClO4 x 4H2O (2), and [(L-H)Pd]ClO4 x 2H2O (3), prepared from L and M2+ salts, the metal is strongly bound by an in-plane N4-coordination (confirmed by X-ray crystal structure determination of la). Formation of trinuclear complexes [(L-H)MsCu2]5+, with two functional Cu2+ ions coordinated to the bidentate sites of L, was evidenced in solution by photometric titration and by isolation of [(L-H)Cu3][PO4][ClO4]2 x 9H2O (4). The trinuclear complexes catalyze the cleavage of RNA-analogue 2-(hydroxypropyl)-p-nitrophenyl phosphate (HPNP), an activated phosphodiester. From a kinetic analysis of the cleavage rate at various HPNP concentrations, parameters KHPNP (the equilibrium constant for binding of HPNP to [(L-H)MsCu2]5+ and kcat (first-order rate constant for cleavage of HPNP when bound to the catalyst) were derived: KM= 170 (Ms= Cu2+), 340 (Ms = Ni2+), 2,600 (Ms = Pd2+) M(-1), kcat = 17 x 10(-3) (Ms= Cu2+) 3.1 x 10(-3) (Ms=Ni2+), 0.22 x 10(-3) (Ms = Pd2+) s(-1). Obviously, the nature of the allosteric metal ion Ms strongly influences both substrate affinity and reactivity of the catalyst [(L-H)MsCu2]5+. Our interpretation of this observation is that subtle differences in the ionic radius of Ms and in its tendency to distort the N4-Ms coordination plane have a significant influence on the conformation of the catalyst (i.e., preorganization of functional Cu2+ ions) and thus on catalytic activity.

Allosteric Regulation↗
PubMed2001

Proteome analysis of Corynebacterium glutamicum.

By the use of different Corynebacterium glutamicum strains more than 1.4 million tons of amino acids, mainly L-glutamate and L-lysine, are produced per year. A project was started recently to elucidate the complete DNA sequence of this bacterium. In this communication we describe an approach to analyze the C. glutamicum proteome, based on this genetic information, by a combination of two-dimensional (2-D) gel electrophoresis and protein identification via microsequencing or mass spectrometry. We used these techniques to resolve proteins of C. glutamicum with the aim to establish 2-D protein maps as a tool for basic microbiology and for strain improvement. In order to analyze the C. glutamicum proteome, methods were established to fractionate the C. glutamicum proteins according to functional entities, i.e., cytoplasm, membranes, and cell wall. Protein spots of the cytoplasmic and membrane fraction were identified by N-terminal sequencing, immunodetection, matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-mass spectrometry (ESI-MS). Additionally, a protocol to analyze proteins secreted by C. glutamicum was established. Approximately 40 protein spots were observed on silver-stained 2-D gels, 12 of which were identified.

Amino Acid Sequence↗
PubMed2001

Carrier-mediated transport of riboflavin in Ashbya gossypii.

The filamentous hemiascomycete Ashbya gossypii is used for industrial riboflavin production. We examined riboflavin uptake and excretion at the plasma membrane using riboflavin auxotrophic and overproducing mutants. The riboflavin uptake system had low activity [Vmax = 20 +/- 4 nmol min(-1) g(-1) mycelial dry weight (dw)] and high affinity (KM = 40 +/- 12 microM). Inhibitor studies with the analogs FMN and FAD revealed high specificity of the uptake system. Excretion of riboflavin was not the consequence of non-specific permeability of the plasma membrane. Excretion rates in the mid-production phase were determined to be 2.5 nmol min(-1) g(-1) dw for wild-type cells and 66.7 nmol min(-1) g(-1) dw for an overproducing mutant, respectively. Inhibition of the reverse reaction, riboflavin uptake, led to an increase in apparent riboflavin efflux in the early production phase, indicating the presence of a separate excretion carrier. Riboflavin accumulation in A. gossypii vacuoles leading to product retention was found to be a secondary transport process. To address the question of whether a flux from the vacuoles back into the cytoplasm is present, we characterized efflux in hyphae in which the plasma membrane was permeabilized with digitonin. Efflux kinetics across the vacuolar membrane were unaffected by the lack of vacuolar H+ATPase activity and ATP, suggesting a passive mechanism. Based on the characterization of riboflavin transport processes in this study, the design of new production strains with improved riboflavin excretion may be possible.

Ascomycota↗
PubMed2001

Deletion of a novel protein kinase with PX and FYVE-related domains increases the rate of differentiation of Trypanosoma brucei.

Growth control of African trypanosomes in the mammalian host is coupled to differentiation of a non-dividing life cycle stage, the stumpy bloodstream form. We show that a protein kinase with novel domain architecture is important for growth regulation. Zinc finger kinase (ZFK) has a kinase domain related to RAC and S6 kinases flanked by a FYVE-related zinc finger and a phox (PX) homology domain. To investigate the function of the kinase during cyclical development, a stable transformation procedure for bloodstream forms of differentiation-competent (pleomorphic) Trypanosoma brucei strains was established. Deletion of both allelic copies of ZFK by homologous recombination resulted in reduced growth of bloodstream-form parasites in culture, which was correlated with an increased rate of differentiation to the non-dividing stumpy form. Growth and differentiation rates were returned to wild-type level by ectopic ZFK expression. The phenotype is stage-specific, as growth of procyclic (insect form) trypanosomes was unaffected, and Deltazfk/Deltazfk clones were able to undergo full cyclical development in the tsetse fly vector. Deletion of ZFK in a differentiation-defective (monomorphic) strain of T. brucei did not change its growth rate in the bloodstream stage. This suggests a function of ZFK associated with the trypanosomes' decision between either cell cycle progression, as slender bloodstream form, or differentiation to the non-dividing stumpy form.

Amino Acid Sequence↗
PubMed2001

Sensing nitrogen limitation in Corynebacterium glutamicum: the role of glnK and glnD.

A novel nitrogen control system regulating the transcription of genes expressed in response to nitrogen starvation in Corynebacterium glutamicum was identified by us recently. In this communication, we also show that the nitrogen regulation cascade in C. glutamicum functions by a new mechanism, although components highly similar to sensor and signal transmitter proteins of Escherichia coli are used, namely uridylyltransferase and a PII-type GlnK protein. The genes encoding these key components of the nitrogen regulation cascade, glnD and glnK, are organized in an operon together with amtB, which codes for an ammonium permease. Using a combination of site-directed mutagenesis, RNA hybridization experiments, reporter gene assays, transport measurements and non-denaturing gel electrophoresis followed by immunodetection, we showed that GlnK is essential for nitrogen control and that signal transduction is transmitted by uridylylation of this protein. As a consequence of the latter, a glnD deletion strain lacking uridylyltransferase is impaired in its response to nitrogen shortage. The glnD mutant revealed a decreased growth rate in the presence of limiting amounts of ammonium or urea; additionally, changes in its protein profile were observed, as shown by in vivo labelling and two-dimensional PAGE. In contrast to E. coli, expression of glnD is upregulated upon nitrogen limitation in C. glutamicum. This indicates that the glnD gene product is probably not the primary sensor of nitrogen status in C. glutamicum as shown for enterobacteria. In accordance with this hypothesis, we found a deregulated nitrogen control as a result of the overexpression of glnD. Furthermore, quantification of cytoplasmic amino acid pools excluded the possibility that a fall in glutamine concentration is perceived as the signal for nitrogen starvation by C. glutamicum, as is found in enterobacteria. Direct measurements of the intracellular ammonium pool indicated that the concentration of this compound might indicate the cellular nitrogen status. Deduced from glnK and glnD expression patterns and the genetic organization of these genes, this regulatory mechanism is also present in Corynebacterium diphtheriae, the causative agent of diphtheria.

Bacterial Proteins↗
PubMed2001

The low-molecular-mass subunit of the cell wall channel of the Gram-positive Corynebacterium glutamicum. Immunological localization, cloning and sequencing of its gene porA.

The 5-kDa protein PorA of the Gram-positive bacterium Corynebacterium glutamicum is the subunit of the cell wall channel. Antibodies raised against PorA specifically detected the protein on the cell surface. PorA was sequenced using Edman degradation and a gas phase sequencer. The primary sequence was used to create degenerate oligonucleotide primers. The gene of the channel-forming protein and its flanking regions were obtained by PCR followed by inverse PCR. The gene porA comprises 138 bp and encodes a 45-amino-acid-long acidic polypeptide with an excess of four negatively charged amino acids in agreement with the high cation selectivity of the PorA cell wall channel. PorA does not contain an N-terminal extension. A ribosomal-binding site was recognized 6 bp before the start codon ATG of porA. It codes for the smallest subunit of a membrane channel known so far and for the first cell wall channel protein of a corynebacterium. Southern blots demonstrated that only the chromosomes of corynebacteria contain homologous sequences to porA; no hybridization could be detected with DNA from other mycolata.

Amino Acid Sequence↗