PubMed Health⌕ Search

Biomedical subjects

Dieter Jahn

Publications and source records attributed to Dieter Jahn.

At least 19 recordsLinked to original sources

Strict Aerobic Lifestyle and Anaerobic Survival of Bacteria: Inseparable Twins?

For many decades the existence of strict aerobic bacteria was part of every textbook. However, considering habitats like soils or surfaces, many of these microorganisms are exposed to drastic changes in oxygen tension. A simple rain shower can change oxygen diffusion rates by a factor of 10.000. Thus, for many of the so-called strict aerobic bacteria, anaerobic growth and survival strategies were discovered, mainly relying on the use of alternative electron acceptors to oxygen, redox-active metabolites, or fermentation processes generating ATP at the substrate level. Survival without growth was recognized as an important lifestyle of bacteria. With the increasing availability of genome data, many highly diverse growth and survival strategies have become apparent in bacteria. But the overall picture is far from complete. Only recently, a novel puzzle piece of the anaerobic survival strategy of the opportunistic pathogen and model bacterium Pseudomonas aeruginosa in the absence of alternative electron acceptors was elucidated. It relies on the re-wiring of carbon flux away from the Entner-Doudoroff pathway towards the pentose-phosphate pathway and use of a phosphoketolase to allow for metabolic flux while preventing nonproductive NADH formation under these fermentation conditions and for ATP generation via acetate kinase.

Anaerobiosis↗

Functional definition of the tobacco protoporphyrinogen IX oxidase substrate-binding site.

PPO (protoporphyrinogen IX oxidase) catalyses the flavin-dependent six-electron oxidation of protogen (protoporphyrinogen IX) to form proto (protoporphyrin IX), a crucial step in haem and chlorophyll biosynthesis. The apparent K(m) value for wild-type tobacco PPO2 (mitochondrial PPO) was 1.17 muM, with a V(max) of 4.27 muM.min(-1).mg(-1) and a catalytic activity k(cat) of 6.0 s(-1). Amino acid residues that appear important for substrate binding in a crystal structure-based model of the substrate docked in the active site were interrogated by site-directed mutagenesis. PPO2 variant F392H did not reveal detectable enzyme activity indicating an important role of Phe(392) in substrate ring A stacking. Mutations of Leu(356), Leu(372) and Arg(98) increased k(cat) values up to 100-fold, indicating that the native residues are not essential for establishing an orientation of the substrate conductive to catalysis. Increased K(m) values of these PPO2 variants from 2- to 100-fold suggest that these residues are involved in, but not essential to, substrate binding via rings B and C. Moreover, one prominent structural constellation of human PPO causing the disease variegate porphyria (N67W/S374D) was successfully transferred into the tobacco PPO2 background. Therefore tobacco PPO2 represents a useful model system for the understanding of the structure-function relationship underlying detrimental human enzyme defects.

Amino Acids↗

Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium.

Production and secretion of a 28,172 Da hydrolase from Thermobifida fusca (TFH) in Bacillus megaterium MS941 and WH323 was investigated in shake flask and pH controlled bioreactors. Successful production of heterologous TFH was achieved by adapting the original tfh gene to the optimal codon usage of B. megaterium. A codon adaption index close to one was reached. The codon optimized tfh was cloned into an open reading frame with DNA sequence for the N-terminal signal peptide of B. megaterium lipase A and a C-terminal His(6)-tag, all under the control of a xylose inducible promoter. Successful TFH production and secretion were observed using batch reactor cultivations with complex medium. Expression of the tfh gene from the P(xylA) promoter and secretion of produced TFH were compared in detail to batch reactor cultivations with semi-defined growth medium. For the first time, significant TFH secretion was achieved using a semi-defined medium in glucose limited fed batch cultivations yielding 10-fold higher cell densities compared to LB medium cultivation. Comparable volumetric TFH activities were obtained for both cultivation strategies. Surprisingly, measured specific TFH activities exhibited drastic discrepancies between preparations from LB and semi-defined medium grown B. megaterium. TFH recovery by Ni-chelate affinity chromatography resulted in higher purification factors when LB medium was used. These results indicated that secreted TFH is favorably produced by batch cultures of B. megaterium WH323 in LB medium.

Actinomycetales↗

Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium.

A multiple vector system for the intracellular high-level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N- and C-terminal fusion of a protein of interest to a Strep II and a His(6)-tag is possible. Corresponding genes are expressed under the control of a xylose-inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N- or C-terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one-step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry.

Bacillus megaterium↗

Production of recombinant antibody fragments in Bacillus megaterium.

BACKGROUND: Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments. RESULTS: The lysozyme specific single chain Fv (scFv) fragment D1.3 was successfully produced using B. megaterium. The impact of culture medium composition, gene expression time and culture temperatures on the production of functional scFv protein was systematically analyzed. A production and secretion at 41 degrees C for 24 h using TB medium was optimal for this individual scFv. Interestingly, these parameters were very different to the optimal conditions for the expression of other proteins in B. megaterium. Per L culture supernatant, more than 400 microg of recombinant His6-tagged antibody fragment were purified by one step affinity chromatography. The material produced by B. megaterium showed an increased specific activity compared to material produced in E. coli. CONCLUSION: High yields of functional scFv antibody fragments can be produced and secreted into the culture medium by B. megaterium, making this production system a reasonable alternative to E. coli.

Journal Article↗

SYSTOMONAS--an integrated database for systems biology analysis of Pseudomonas.

To provide an integrated bioinformatics platform for a systems biology approach to the biology of pseudomonads in infection and biotechnology the database SYSTOMONAS (SYSTems biology of pseudOMONAS) was established. Besides our own experimental metabolome, proteome and transcriptome data, various additional predictions of cellular processes, such as gene-regulatory networks were stored. Reconstruction of metabolic networks in SYSTOMONAS was achieved via comparative genomics. Broad data integration is realized using SOAP interfaces for the well established databases BRENDA, KEGG and PRODORIC. Several tools for the analysis of stored data and for the visualization of the corresponding results are provided, enabling a quick understanding of metabolic pathways, genomic arrangements or promoter structures of interest. The focus of SYSTOMONAS is on pseudomonads and in particular Pseudomonas aeruginosa, an opportunistic human pathogen. With this database we would like to encourage the Pseudomonas community to elucidate cellular processes of interest using an integrated systems biology strategy. The database is accessible at http://www.systomonas.de.

Bacterial Proteins↗

High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium.

BACKGROUND: During the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Here, recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA) which is used in the reverse synthesis of beta-lactam antibiotics were systematically improved. RESULTS: For this purpose, the PGA leader peptide was replaced by the B. megaterium LipA counterpart. A production strain deficient in the extracellular protease NprM and in xylose utilization to prevent gene inducer deprivation was constructed and employed. A buffered mineral medium containing calcium ions and defined amino acid supplements for optimal PGA production was developed in microscale cultivations and scaled up to a 2 Liter bioreactor. Productivities of up to 40 mg PGA per L growth medium were reached. CONCLUSION: The combination of genetic and medium optimization led to an overall 7-fold improvement of PGA production and export in B. megaterium. The exclusion of certain amino acids from the minimal medium led for the first time to higher volumetric PGA activities than obtained for complex medium cultivations.

Journal Article↗

MetaQuant: a tool for the automatic quantification of GC/MS-based metabolome data.

UNLABELLED: MetaQuant is a Java-based program for the automatic and accurate quantification of GC/MS-based metabolome data. In contrast to other programs MetaQuant is able to quantify hundreds of substances simultaneously with minimal manual intervention. The integration of a self-acting calibration function allows the parallel and fast calibration for several metabolites simultaneously. Finally, MetaQuant is able to import GC/MS data in the common NetCDF format and to export the results of the quantification into Systems Biology Markup Language (SBML), Comma Separated Values (CSV) or Microsoft Excel (XLS) format. AVAILABILITY: MetaQuant is written in Java and is available under an open source license. Precompiled packages for the installation on Windows or Linux operating systems are freely available for download. The source code as well as the installation packages are available at http://bioinformatics.org/metaquant

Algorithms↗

Heme biosynthesis in Methanosarcina barkeri via a pathway involving two methylation reactions.

The methanogenic archaeon Methanosarcina barkeri synthesizes protoheme via precorrin-2, which is formed from uroporphyrinogen III in two consecutive methylation reactions utilizing S-adenosyl-L-methionine. The existence of this pathway, previously exclusively found in the sulfate-reducing delta-proteobacterium Desulfovibrio vulgaris, was demonstrated for M. barkeri via the incorporation of two methyl groups from methionine into protoheme.

Archaeal Proteins↗

JVirGel 2.0: computational prediction of proteomes separated via two-dimensional gel electrophoresis under consideration of membrane and secreted proteins.

MOTIVATION: After the publication of JVirGel 1.0 in 2003 we got many requests and suggestions from the proteomics community to further improve the performance of the software and to add additional useful new features. RESULTS: The integration of the PrediSi algorithm for the prediction of signal peptides for the Sec-dependent protein export into JVirGel 2.0 allows the exclusion of most exported preproteins from calculated proteomic maps and provides the basis for the calculation of Sec-based secretomes. A tool for the identification of transmembrane helices carrying proteins (JCaMelix) and the prediction of the corresponding membrane proteome was added. Finally, in order to directly compare experimental and calculated proteome data, a function to overlay and evaluate predicted and experimental two-dimensional gels was included. AVAILABILITY: JVirGel 2.0 is freely available as precompiled package for the installation on Windows or Linux operating systems. Furthermore, there is a completely platform-independent Java version available for download. Additionally, we provide a Java Server Pages based version of JVirGel 2.0 which can be operated in nearly all web browsers. All versions are accessible at http://www.jvirgel.de

Algorithms↗

Crystal structure of a non-discriminating glutamyl-tRNA synthetase.

Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA(Glu) and tRNA(Gln) with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA(Glu). Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS(Tel)) in complex with glutamate at a resolution of 2.45 A. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS(Tth)). We confirm experimentally that GluRS(Tel) is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA(Glu) and of Glu-tRNA(Gln). Anticodons of tRNA(Glu) (34C/UUC36) and tRNA(Gln) (34C/UUG36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS(Tth) by the residue Arg358. In ND-GluRS(Tel) this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA(Gln) to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity.

Amino Acid Sequence↗

JProGO: a novel tool for the functional interpretation of prokaryotic microarray data using Gene Ontology information.

A novel program suite was implemented for the functional interpretation of high-throughput gene expression data based on the identification of Gene Ontology (GO) nodes. The focus of the analysis lies on the interpretation of microarray data from prokaryotes. The three well established statistical methods of the threshold value-based Fisher's exact test, as well as the threshold value-independent Kolmogorov-Smirnov and Student's t-test were employed in order to identify the groups of genes with a significantly altered expression profile. Furthermore, we provide the application of the rank-based unpaired Wilcoxon's test for a GO-based microarray data interpretation. Further features of the program include recognition of the alternative gene names and the correction for multiple testing. Obtained results are visualized interactively both as a table and as a GO subgraph including all significant nodes. Currently, JProGO enables the analysis of microarray data from more than 20 different prokaryotic species, including all important model organisms, and thus constitutes a useful web service for the microbial research community. JProGO is freely accessible via the web at the following address: http://www.jprogo.de.

Archaea↗

The substrate radical of Escherichia coli oxygen-independent coproporphyrinogen III oxidase HemN.

During porphyrin biosynthesis the oxygen-independent coproporphyrinogen III oxidase (HemN) catalyzes the oxidative decarboxylation of the propionate side chains of rings A and B of coproporphyrinogen III to form protoporphyrinogen IX. The enzyme utilizes a 5'-deoxyadenosyl radical to initiate the decarboxylation reaction, and it has been proposed that this occurs by stereo-specific abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chains leading to a substrate radical. Here we provide EPR-spectroscopic evidence for intermediacy of the latter radical by observation of an organic radical EPR signal in reduced HemN upon addition of S-adenosyl-L-methionine and the substrate coproporphyrinogen III. This signal (g(av) = 2.0029) shows a complex pattern of well resolved hyperfine splittings from at least five different hydrogen atoms. The radical was characterized using regiospecifically labeled (deuterium or 15N) coproporphyrinogen III molecules. They had been generated from a multienzyme mixture and served as efficient substrates. Reaction of HemN with coproporphyrinogen III, perdeuterated except for the methyl groups, led to the complete loss of resolved proton hyperfine splittings. Substrates in which the hydrogens at both alpha- and beta-positions, or only at the beta-positions of the propionate side chains, or those of the methylene bridges, were deuterated showed that there is coupling with hydrogens at the alpha-, beta-, and methylene bridge positions. Deuterium or 15N labeling of the pyrrole nitrogens without labeling the side chains only led to a slight sharpening of the radical signal. Together, these observations clearly identified the radical signal as substrate-derived and indicated that, upon abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chain by the 5'-deoxyadenosyl radical, a comparatively stable delocalized substrate radical intermediate is formed in the absence of electron acceptors. The observed hyperfine constants and g values show that this coproporphyrinogenyl radical is allylic and encompasses carbon atoms 3', 3, and 4.

Bacillus megaterium↗

A protein database constructed from low-coverage genomic sequence of Bacillus megaterium and its use for accelerated proteomic analysis.

Peptide mass fingerprint (PMF) matching is a high-throughput method used for protein spot identification in connection with two-dimensional gel electrophoresis (2DE). However, the success of PMF matching largely depends on whether the proteins to be identified exist in the database searched. Consequently, it is often necessary to apply other more sophisticated but also time-consuming technologies to generate sequence-tags for definitive protein identification. On the other hand, modern sequencing technologies are generating a large quantity of DNA sequences, first in unfinished form or with low genome coverage due to the time-consuming and thus limiting steps of finishing and annotation. We recently started to sequence the genome of Bacillus megaterium DSM 319, a bacterium of industrial interest. In this study, we demonstrate that a protein database generated from merely three-fold coverage, unfinished genomic sequences of this bacterium allows a fast and reliable protein spot identification solely based on PMF from high-throughput MALDI-TOF MS analysis. We further show that the strain-specific protein database from low coverage genomic sequence greatly outperforms the commonly used cross-species databases constructed from 13 completely sequenced Bacillus strains for protein spot identification via PMF.

Algorithms↗

Evolutionary relationship between initial enzymes of tetrapyrrole biosynthesis.

Glutamate-1-semialdehyde 2,1-aminomutase (GSAM) is the second enzyme in the C(5) pathway of tetrapyrrole biosynthesis found in most bacteria, in archaea and in plants. It catalyzes the transamination of glutamate-1-semialdehyde to 5-aminolevulinic acid (ALA) in a pyridoxal 5'-phosphate (PLP)-dependent manner. We present the crystal structure of GSAM from the thermophilic cyanobacterium Thermosynechococcus elongatus (GSAM(Tel)) in its PLP-bound form at 2.85A resolution. GSAM(Tel) is a symmetric homodimer, whereas GSAM from Synechococcus (GSAM(Syn)) has been described as asymmetric. The symmetry of GSAM(Tel) thus challenges the previously proposed negative cooperativity between monomers of this enzyme. Furthermore, GSAM(Tel) reveals an extensive flexible region at the interface of the proposed complex of GSAM with glutamyl-tRNA reductase (GluTR), the preceding enzyme in tetrapyrrole biosynthesis. Compared to GSAM(Syn), the monomers of GSAM(Tel) are rotated away from each other along the dimerization interface by 10 degrees . The associated flexibility of GSAM may be essential for complex formation with GluTR to occur. Unexpectedly, we find that GSAM is structurally related to 5-aminolevulinate synthase (ALAS), the ALA-producing enzyme in the Shemin pathway of alpha-proteobacteria and non-plant eukaryotes. This structural relationship applies also to the corresponding subfamilies of PLP-dependent enzymes. We thus propose that the CoA-subfamily (including ALAS) and the aminotransferase subfamily II (including GSAM) are evolutionarily closely related and that ALAS may thus have evolved from GSAM.

5-Aminolevulinate Synthetase↗

Bacillus subtilis Fnr senses oxygen via a [4Fe-4S] cluster coordinated by three cysteine residues without change in the oligomeric state.

The oxygen regulator Fnr is part of the regulatory cascade in Bacillus subtilis for the adaptation to anaerobic growth conditions. In vivo complementation experiments revealed the essential role of only three cysteine residues (C227, C230, C235) at the C-terminus of B. subtilis Fnr for the transcriptional activation of the nitrate reductase operon (narGHJI) and nitrite extrusion protein gene (narK) promoters. UV/VIS, electron paramagnetic spin resonance (EPR) and Mössbauer spectroscopy experiments in combination with iron and sulphide content determinations using anaerobically purified recombinant B. subtilis Fnr identified the role of these three cysteine residues in the formation of one [4Fe-4S]2+ cluster per Fnr molecule. The obtained Mössbauer parameters are supportive for a [4Fe-4S]2+ cluster with three cysteine ligated iron sites and one non-cysteine ligated iron site. Gel filtration experiments revealed a stable dimeric structure for B. subtilis Fnr which is independent of the presence of the [4Fe-4S]2+ cluster. Gel mobility shift and in vitro transcription assays demonstrated the essential role of an intact [4Fe-4S]2+ cluster for promoter binding and transcriptional activation. An amino acid exchange introduced in the proposed alphaD-helix of B. subtilis Fnr (G149S) abolished its in vivo and in vitro activities indicating its importance for intramolecular signal transduction. The clear differences in the localization and coordination of the [4Fe-4S] cluster and in the organization of the oligomeric state between Escherichia coli and B. subtilis Fnr indicate differences in their mode of action.

Amino Acid Sequence↗

The Bacillus subtilis nrdEF genes, encoding a class Ib ribonucleotide reductase, are essential for aerobic and anaerobic growth.

Ribonucleotide reductases (RNRs) are essential for the biosynthesis of the deoxyribonucleoside triphosphates of DNA. Recently, it was proposed that externally supplied deoxyribonucleosides or DNA is required for the growth of Bacillus subtilis under strict anaerobic conditions (M. J. Folmsbee, M. J. McInerney, and D. P. Nagle, Appl. Environ. Microbiol. 70:5252-5257, 2004). Cultivation of B. subtilis on minimal medium in the presence of oxygen indicators in combination with oxygen electrode measurements and viable cell counting demonstrated that growth occurred under strict anaerobic conditions in the absence of externally supplied deoxyribonucleosides. The nrdEF genes encode the only obvious RNR in B. subtilis. A temperature-sensitive nrdE mutant failed to grow under aerobic and anaerobic conditions, indicating that this oxygen-dependent class I RNR has an essential role under both growth conditions. Aerobic growth and anaerobic growth of the nrdE mutant were rescued by addition of deoxynucleotides. The nrd locus consists of an nrdI-nrdE-nrdF-ymaB operon. The 5' end of the corresponding mRNA revealed transcriptional start sites 45 and 48 bp upstream of the translational start of nrdI. Anaerobic transcription of the operon was found to be dependent on the presence of intact genes for the ResDE two-component redox regulatory system. Two potential ResD binding sites were identified approximately 62 bp (site A) and 50 bp (site B) upstream of the transcriptional start sites by a bioinformatic approach. Only mutation of site B eliminated nrd expression. Aerobic transcription was ResDE independent but required additional promoter elements localized between 88 and 275 bp upstream of the transcriptional start.

Aerobiosis↗

A Bacillus megaterium plasmid system for the production, export, and one-step purification of affinity-tagged heterologous levansucrase from growth medium.

A multiple vector system for the production and export of recombinant affinity-tagged proteins in Bacillus megaterium was developed. Up to 1 mg/liter of a His6-tagged or Strep-tagged Lactobacillus reuteri levansucrase was directed into the growth medium, using the B. megaterium esterase LipA signal peptide, and recovered by one-step affinity chromatography.

Affinity Labels↗