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M Bott

Publications and source records attributed to M Bott.

At least 19 recordsLinked to original sources
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

A high-resolution reference map for cytoplasmic and membrane-associated proteins of Corynebacterium glutamicum.

We present a high-resolution reference map for soluble proteins obtained from Corynebacterium glutamicum cells grown in glucose minimal medium. The analysis window covers the pl range from 4-6 and the molecular mass range from 5-100 kDa. Using overlapping narrow immobilized pH gradients for isoelectric focusing, 970 protein spots were detected after second-dimensional separation on SDS-polyacrylamide gels and colloidal Coomassie-staining. By tryptic peptide mass fingerprinting 169 protein spots were identified, representing 152 different proteins including many enzymes involved in central metabolism (18), amino acid biosynthesis (24) and nucleotide biosynthesis (11). Thirty-five of the identified proteins have no known function. A comparison of the observed and the expected physicochemical properties of the identified proteins indicated that nine proteins were covalently modified, since variants with apparently identical molecular mass, but differing pl were detected. The N-termini of eight proteins were determined by post-source decay (PSD) analysis of selected peptides. In addition to the soluble proteins, a map of the membrane-bound proteins within the pl range 4-7 is presented, which contains 660 protein spots, 22 of which were identified, representing 13 different proteins.

Bacterial Proteins↗
PubMed2001

Molecular analysis of the cytochrome bc1-aa3 branch of the Corynebacterium glutamicum respiratory chain containing an unusual diheme cytochrome c1.

In this work, the genes for cytochrome aa3 oxidase and the cytochrome bc1 complex in the gram-positive soil bacterium Corynebacterium glutamicum were identified. The monocistronic ctaD gene encoded a 65-kDa protein with all features typical for subunit I of cytochrome aa3 oxidases. A ctaD deletion mutant lacked the characteristic 600 nm peak in redox difference spectra, and growth in glucose minimal medium was strongly impaired. The genes encoding subunit III of cytochrome aa3 (ctaE) and the three characteristic subunits of the cytochrome bc1 complex (qcrABC) were clustered in the order ctaE-qcrCAB. Analysis of the deduced primary structures revealed a number of unusual features: (1) cytochrome c1 (QcrC, 30 kDa) contained two Cys-X-X-Cys-His motifs for covalent heme attachment, indicating that it is a diheme c-type cytochrome; (2) the 'Rieske' iron-sulphur protein (QcrA, 45 kDa) contained three putative transmembrane helices in the N-terminal region rather than only one; and (3) cytochrome b (QcrB, 60 kDa) contained, in addition to the conserved part with eight transmembrane helices, a C-terminal extension of about 120 amino acids, which presumably is located in the cytoplasm. Staining of C. glutamicum proteins for covalently bound heme indicated the presence of a single, membrane-bound c-type cytochrome with an apparent molecular mass of about 31 kDa. Since this protein was missing in a qcrCAB deletion mutant, it most likely corresponds to cytochrome c1. Similar to the deltactaD mutant, the deltaqcrCAB mutant showed strongly impaired growth in glucose minimal medium, which indicates that the bc1-aa3 pathway is the main route of respiration under these conditions.

Amino Acid Sequence↗
PubMed2001

Catabolite repression of the citrate fermentation genes in Klebsiella pneumoniae: evidence for involvement of the cyclic AMP receptor protein.

Klebsiella pneumoniae is able to grow anaerobically with citrate as a sole carbon and energy source by a fermentative pathway involving the Na(+)-dependent citrate carrier CitS, citrate lyase, and oxaloacetate decarboxylase. The corresponding genes are organized in the divergent citC and citS operons, whose expression is strictly dependent on the citrate-sensing CitA-CitB two-component system. Evidence is provided here that the citrate fermentation genes are subject to catabolite repression, since anaerobic cultivation with a mixture of citrate and glucose or citrate and gluconate resulted in diauxic growth. Glucose, gluconate, and also glycerol decreased the expression of a chromosomal citS-lacZ fusion by 60 to 75%, whereas a direct inhibition of the citrate fermentation enzymes was not observed. The purified cyclic AMP (cAMP) receptor protein (CRP) of K. pneumoniae bound to two sites in the citC-citS intergenic region, which were centered at position -41.5 upstream of the citC and citS transcriptional start sites. Binding was apparently stimulated by the response regulator CitB. These data indicate that catabolite repression of the citrate fermentation genes is exerted by CRP and that in the absence of repressing carbon sources the cAMP-CRP complex serves to enhance the basal, CitB-dependent transcription level.

Anaerobiosis↗
PubMed2000

Identification of triphosphoribosyl-dephospho-CoA as precursor of the citrate lyase prosthetic group.

The gamma-subunit of citrate lyase (EC 4.1.3.6) contains the prosthetic group 2'-(5"-phosphoribosyl)-3'-dephospho-CoA and serves as an acyl carrier protein (ACP). We recently showed that in Escherichia coli the proteins CitG and CitX are essential for holo-ACP synthesis and provided evidence that CitG catalyzes the formation of a prosthetic group precursor from ATP and dephospho-CoA, which is subsequently attached via phosphodiester linkage to apo-ACP by CitX. Here we prove that CitG indeed catalyzes the conversion of ATP and dephospho-CoA to adenine and 2'-(5"-triphosphoribosyl)-3'-dephospho-CoA, the predicted precursor of the prosthetic group. Furthermore, this precursor was transferred by CitX to apo-ACP, yielding holo-ACP. Thus, our proposed mechanism for holo-ACP synthesis could be verified.

ATP Citrate (pro-S)-Lyase↗
PubMed2000

Biosynthesis of the prosthetic group of citrate lyase.

Citrate lyase (EC 4.1.3.6) catalyzes the cleavage of citrate to acetate and oxaloacetate and is composed of three subunits (alpha, beta, and gamma). The gamma-subunit serves as an acyl carrier protein (ACP) and contains the prosthetic group 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA, which is attached via a phosphodiester linkage to serine-14 in the enzyme from Klebsiella pneumoniae. In this work, we demonstrate by genetic and biochemical studies with citrate lyase of Escherichia coli and K. pneumoniae that the conversion of apo-ACP into holo-ACP is dependent on the two proteins, CitX (20 kDa) and CitG (33 kDa). In the absence of CitX, only apo-ACP was synthesized in vivo, whereas in the absence of CitG, an adenylylated ACP was produced, with the AMP residue attached to serine-14. The adenylyltransferase activity of CitX could be verified in vitro with purified CitX and apo-ACP plus ATP as substrates. Besides ATP, CTP, GTP, and UTP also served as nucleotidyl donors in vitro, showing that CitX functions as a nucleotidyltransferase. The conversion of apo-ACP into holo-ACP was achieved in vitro by incubation of apo-ACP with CitX, CitG, ATP, and dephospho-CoA. ATP could not be substituted with GTP, CTP, UTP, ADP, or AMP. In the absence of CitG or dephospho-CoA, AMP-ACP was formed. Remarkably, it was not possible to further convert AMP-ACP to holo-ACP by subsequent incubation with CitG and dephospho-CoA. This demonstrates that AMP-ACP is not an intermediate during the conversion of apo- into holo-ACP, but results from a side activity of CitX that becomes effective in the absence of its natural substrate. Our results indicate that holo-ACP formation proceeds as follows. First, a prosthetic group precursor [presumably 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA] is formed from ATP and dephospho-CoA in a reaction catalyzed by CitG. Second, holo-ACP is formed from apo-ACP and the prosthetic group precursor in a reaction catalyzed by CitX.

Acyl Carrier Protein↗
PubMed1999

The periplasmic domain of the histidine autokinase CitA functions as a highly specific citrate receptor.

The two-component regulatory system CitA/CitB is essential for induction of the citrate fermentation genes in Klebsiella pneumoniae. CitA represents a membrane-bound sensor kinase consisting of a periplasmic domain flanked by two transmembrane helices, a linker domain and the conserved kinase or transmitter domain. A fusion protein (MalE-CitAC) composed of the maltose-binding protein and the CitA kinase domain (amino acids 327-547) showed constitutive autokinase activity and transferred the gamma-phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of CitB was inhibited by a D56N exchange, indicating that H-350 and D-56 represent the phosphorylation sites of CitA and CitB respectively. In the presence of ATP, CitB-D56N formed a stable complex with MalE-CitAC. To analyse the sensory properties of CitA, the periplasmic domain (amino acids 45-176) was overproduced as a soluble, cytoplasmic protein with a C-terminally attached histidine tag (CitAPHis). Purified CitAPHis bound citrate, but none of the other tri- and dicarboxylates tested, with high affinity (KD approximately 5 microM at pH 7) in a 1:1 stoichiometry. As shown by isothermal titration calorimetry, the binding reaction was driven by the enthalpy change (DeltaH = -76.3 kJ mol-1), whereas the entropy change was opposed (-TDeltaS = + 46.3 kJ mol-1). The pH dependency of the binding reaction indicated that the dianionic form H-citrate2- is the citrate species recognized by CitAPHis. In the presence of Mg2+ ions, the dissociation constant increased significantly, suggesting that the Mg-citrate complex is not bound by CitAPHis. This work defines the periplasmic domain of CitA as a highly specific citrate receptor and elucidates the binding characteristics of CitAPHis.

Bacterial Proteins↗
PubMed1999

A membrane-bound NAD(P)+-reducing hydrogenase provides reduced pyridine nucleotides during citrate fermentation by Klebsiella pneumoniae.

During anaerobic growth of Klebsiella pneumoniae on citrate, 9.4 mmol of H2/mol of citrate (4-kPa partial pressure) was formed at the end of growth besides acetate, formate, and CO2. Upon addition of NiCl2 (36 microM) to the growth medium, hydrogen formation increased about 36% to 14.8 mmol/mol of citrate (6 kPa), and the cell yield increased about 15%. Cells that had been harvested and washed under anoxic conditions exhibited an H2-dependent formation of NAD(P)H in vivo. The reduction of internal NAD(P)+ was also achieved by the addition of formate. In crude extracts, the H2:NAD+ oxidoreductase activity was 0.13 micromol min-1 mg-1, and 76% of this activity was found in the washed membrane fraction. The highest specific activities of the membrane fraction were observed in 50 mM potassium phosphate, with 1.6 micromol of NADPH formed min-1 mg-1 at pH 7.0 and 1.7 micromol of NADH formed min-1 mg-1 at pH 9.5. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the Na+/H+ antiporter monensin, the H2-dependent reduction of NAD+ by membrane vesicles decreased only slightly (about 16%). The NADP+- or NAD+-reducing hydrogenases were solubilized from the membranes with the detergent lauryldimethylamine-N-oxide or Triton X-100. NAD(P)H formation with H2 as electron donor, therefore, does not depend on an energized state of the membrane. It is proposed that hydrogen which is formed by K. pneumoniae during citrate fermentation is recaptured by a novel membrane-bound, oxygen-sensitive H2:NAD(P)+ oxidoreductase that provides reducing equivalents for the synthesis of cell material.

Anaerobiosis↗
PubMed1998

The Escherichia coli citrate carrier CitT: a member of a novel eubacterial transporter family related to the 2-oxoglutarate/malate translocator from spinach chloroplasts.

Under anoxic conditions in the presence of an oxidizable cosubstrate such as glucose or glycerol, Escherichia coli converts citrate to acetate and succinate. Two enzymes are specifically required for the fermentation of the tricarboxylic acid, i.e., a citrate uptake system and citrate lyase. Here we report that the open reading frame (designated citT) located at 13.90 min on the E. coli chromosome between rna and the citrate lyase genes encodes a citrate carrier. E. coli transformed with a plasmid expressing citT was capable of aerobic growth on citrate, which provides convincing evidence for a function of CitT as a citrate carrier. Transport studies with cell suspensions of the transformed strain indicated that CitT catalyzes a homologous exchange of citrate or a heterologous exchange against succinate, fumarate, or tartrate. Since succinate is the end product of citrate fermentation in E. coli, it is likely that CitT functions in vivo as a citrate/succinate antiporter. Analysis of the primary sequence showed that CitT (487 amino acids, 53.1 kDa) is a highly hydrophobic protein with 12 putative transmembrane helices. Sequence comparisons revealed that CitT is related to the 2-oxoglutarate/malate translocator (SODiT1 gene product) from spinach chloroplasts and five bacterial gene products, none of which has yet been functionally characterized. It is suggested that the E. coli CitT protein is a member of a novel family of eubacterial transporters involved in the transport of di- and tricarboxylic acids.

Amino Acid Sequence↗
PubMed1997

Methylmalonyl-CoA decarboxylase from Propionigenium modestum--cloning and sequencing of the structural genes and purification of the enzyme complex.

Methylmalonyl-CoA decarboxylase catalyses the only energy-conserving step during succinate fermentation by Propionigenium modestum: the decarboxylation of (S)-methylmalonyl-CoA to propionyl-CoA is coupled to the vectorial transport of Na+ across the cytoplasmic membrane, thereby creating a sodium ion motive force that is used for ATP synthesis. By taking advantage of the sequence similarity between the beta-subunits of other Na+-transport decarboxylases, a portion of the P. modestum beta-subunit gene was amplified by PCR with degenerated primers. The cloned PCR product then served as homologous probe for cloning suitable fragments from genomic DNA. Sequence analysis of a 3.7-kb region identified four genes which probably form a transcriptional unit, mmdADCB. Remarkably, a mmdE gene which is present in the homologous mmdADECB cluster from Veillonella parvula and encodes the 6-kDa epsilon-subunit, is missing in P. modestum. By sequence comparisons, the following functions could be assigned to the P. modestum proteins: MmdA (56.1 kDa; alpha-subunit), carboxyltransferase; MmdB (41.2 kDa; beta-subunit), carboxybiotin-carrier-protein decarboxylase; MmdC (13.1 kDa; gamma-subunit), biotin carrier protein. MmdD (14.2 kDa; delta-subunit) presumably is essential for the assembly of the complex, as shown for the corresponding V. parvula protein. Methylmalonyl-CoA decarboxylase was solubilized from membranes of P. modestum with n-dodecylmaltoside and enriched 15-fold by affinity chromatography on monomeric avidin resin. The purified protein was composed of four subunits, three of which were identified by N-terminal sequence analysis as MmdA, MmdD, and MmdC. The purified enzyme exhibited a specific activity of up to 25 U/mg protein and an apparent Km value for (S)-methylmalonyl-CoA of approximately 12 microM. Compared to the five-subunit complex of V. parvula, the four-subunit enzyme of P. modestum appeared to be more labile, presumably a consequence of the lack of the epsilon-subunit.

Amino Acid Sequence↗
PubMed1997

In vitro binding of the response regulator CitB and of its carboxy-terminal domain to A + T-rich DNA target sequences in the control region of the divergent citC and citS operons of Klebsiella pneumoniae.

The genes specifically required for citrate fermentation in Klebsiella pneumoniae form a cluster on the chromosome consisting of two divergently transcribed groups, citCDEFG and citS-oadGAB-citAB. Northern blot analyses described here and elsewhere indicate that each group forms an operon. The transcriptional start sites of citC and citS, which were mapped in this work by primer extension, are separated by a stretch of 193 bp with an extraordinary high A + T content of 67%. Expression of the citrate fermentation genes was recently shown to be positively controlled by a two-component signal transduction system encoded by the promoter-distal genes of the citS operon, citA (sensor kinase) and citB (response regulator). As a first step towards the functional characterization of CitB, we analysed its DNA-binding properties. To this end, the entire CitB, its N-terminal receiver domain (CitBN), and its C-terminal output domain (CitBC), all modified by a (His)6-tag, were purified. CitB(His) and CitBN(His) could be phosphorylated either with acetylphosphate or with ATP plus MalE-CitAC. The latter protein contains the kinase domain of CitA fused to the C terminus of the maltose-binding protein. Upon phosphorylation, CitB(His) became more resistant towards limited proteolysis by trypsin, reflecting substantial changes in tertiary structure. In gel retardation assays, CitB(His) bound specifically to the citC-citS intergenic region. The retardation pattern changed significantly upon phosphorylation and the apparent binding affinity increased 10 to 100-fold. Depending on the protein concentration, four different phospho-CitB(His)-DNA complexes could be resolved, suggesting the presence of multiple binding sites between citC and citS. DNase I footprints revealed two protected regions extending maximally from -55 to -89 relative to the citS transcription start and from -50 to -96 relative to the citC transcription start. Gel retardation and DNase I footprint assays with CitBC(His) showed that the C-terminal domain is sufficient for specific DNA binding. Since its properties were similar to that of unphosphorylated CitB(His), an essential role of the N-terminal receiver domain in high-affinity DNA binding was indicated. The positions of the binding sites for CitB and of putative recognition sequences for the cAMP receptor protein suggested a model for the interaction of these activators with RNA polymerase.

Bacterial Proteins↗
PubMed1997

Embedded nursing practice: a case study.

Nurses' utilization of case study analysis often focuses on medically-based knowledge which is intertwined within our daily practice. Reflection on nurse-patient interactions with nursing theory as the analytic tool is infrequently undertaken. The case study analysis presented within this paper analyzes an exemplar utilizing the theoretical works of Patricia Benner.

Critical Care↗
PubMed1995

Shared governance. From vision to reality.

Quality patient care is best provided by competent clinical staff members who are committed to their work, organizational values, and goals, and who maintain employment over time. Shared governance has provided the framework for clinical nurses to become more committed to their practice through decision making and to assume accountability for the outcomes of their decisions. At a tertiary teaching facility, shared governance resulted in increased job satisfaction, commitment, and retention among staff nurses. The authors describe the journey from creation of the shared governance vision to reality, and the impact of the changes.

Adult↗
PubMed1995

Regulation of anaerobic citrate metabolism in Klebsiella pneumoniae.

Three enzymes are specifically required for uptake and catabolism of citrate by Klebsiella pneumoniae under anaerobic conditions: a Na+ -dependent citrate carrier (CitS), citrate lyase (CitDEF), and the Na+ pump oxaloacetate decarboxylase (OadGAB). The corresponding genes are clustered on the chromosome, with the citCDEFG genes located upstream and divergent to the citS-oadGAB genes. We found that expression of citS from its native promoter in Escherichia coli requires the DNA region downstream of oadB. Nucleotide sequence analysis of this region revealed the presence of two adjacent genes, citA and citB. By sequence similarity, the predicted CitA and CitB proteins were identified as members of the two-component regulatory systems. The sensor kinase CitA contained, in the N-terminal half, two putative transmembrane helices which enclosed a presumably periplasmic domain of about 130 amino acids. The C-terminal half of the response regulator CitB harboured a helix-turn-helix motif typical of DNA-binding proteins. K. pneumoniae citB null mutants were unable to grow anaerobically with citrate as the sole carbon and energy source (Cit- phenotype). When cultivated anaerobically with citrate plus glycerol, all of the citrate-specific fermentation enzymes were synthesized in the wild type, but not in the citB mutants. This showed that citS, oadGAB and citDEF required the CitB protein for expression and therefore are part of a regulon. In the wild type, synthesis of CitS, oxaloacetate decarboxylase and citrate lyase was dependent on the presence of citrate, sodium ions and a low oxygen tension. In a citA null mutant which expressed citB constitutively at high levels, none of these signals was required for the formation of the citrate fermentation enzymes. This result suggested that citrate, Na+, and oxygen exerted their regulatory effects via the CitA/CitB system. In the presence of these signals, the citAB gene products induced their own synthesis. The positive autoregulation occurred via co-transcription of citAB with citS and oadGAB.

Aconitate Hydratase↗
PubMed1995

Bradyrhizobium japonicum cytochrome c550 is required for nitrate respiration but not for symbiotic nitrogen fixation.

Bradyrhizobium japonicum possesses three soluble c-type cytochromes, c550, c552, and c555. The genes for cytochromes c552 (cycB) and c555 (cycC) were characterized previously. Here we report the cloning, sequencing, and mutational analysis of the cytochrome c550 gene (cycA). A B. japonicum mutant with an insertion in cycA failed to synthesize a 12-kDa c-type cytochrome. This protein was detectable in the cycA mutant complemented with cloned cycA, which proves that it is the cycA gene product. The cycA mutant, a cycB-cycC double mutant, and a cycA-cycB-cycC triple mutant elicited N2-fixing root nodules on soybean (Nod+ Fix+ phenotype); hence, none of these three cytochromes c is essential for respiration supporting symbiotic N2 fixation. However, cytochrome c550, in contrast to cytochromes c552 and c555, was shown to be essential for anaerobic growth of B. japonicum, using nitrate as the terminal electron acceptor.

Amino Acid Sequence↗
PubMed1994

Purification of two active fusion proteins of the Na(+)-dependent citrate carrier of Klebsiella pneumoniae.

The sodium-ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) was purified by means of bioengineerical methods. By fusing the biotin acceptor domain of the alpha-subunit of the oxaloacetate decarboxylase of K. pneumoniae to the C-terminus of CitS, purification of the carrier was achieved by use of a monomeric avidin-Sepharose column. Additionally, we were able to purify a CitS-protein with an N-terminal histidine-tag by immobilized metal chelate affinity chromatography (with Ni2(+)-nitrilotriacetic acid-(NTA-) resin). Both purified fusion proteins showed citrate transport activity after reconstitution into liposomes by the freeze/thaw/sonication procedure.

Bacterial Proteins↗