Biomedical subjects

Jörn Kalinowski

Publications and source records attributed to Jörn Kalinowski.

At least 19 recordsLinked to original sources

PubMed2004

Classification of hyper-variable Corynebacterium glutamicum surface-layer proteins by sequence analyses and atomic force microscopy.

The structural S-layer proteins of 28 different Corynebacterium glutamicum isolates have been analyzed systematically. Treatment of whole C. glutamicum cells with detergents resulted in the isolation of S-layer proteins with different apparent molecular masses, ranging in size from 55 to 66 kDa. The S-layer genes analyzed were characterized by coding regions ranging from 1,473 to 1,533 nucleotides coding for S-layer proteins with a size of 490-510 amino acids. Using PCR techniques, the corresponding S-layer genes of the 28 C. glutamicum isolates were all cloned and sequenced. The deduced amino acid sequences of the S-layer proteins showed identities between 69 and 98% and could be grouped into five phylogenetic classes. Furthermore, sequence analyses indicated that the S-layer proteins of the analyzed C. glutamicum isolates exhibit a mosaic structure of highly conserved and highly variable regions. Several conserved regions were assumed to play a key role in the formation of the C. glutamicum S-layers. Especially the N-terminal signal peptides and the C-terminal anchor sequences of the S-layer proteins showed a nearly perfect amino acid sequence conservation. Analyses by atomic force microscopy revealed a committed hexagonal structure. Morphological diversity of the C. glutamicum S-layers was observed in a class-specific unit cell dimension (ranging from 15.2 to 17.4 nm), which correlates with the sequence similarity-based classification. It could be demonstrated that differences in the primary structure of the S-layer proteins were reflected by the S-layer morphology.

Amino Acid Sequence↗

PubMed2004

The glycosylated cell surface protein Rpf2, containing a resuscitation-promoting factor motif, is involved in intercellular communication of Corynebacterium glutamicum.

The genome of Corynebacterium glutamicum ATCC 13032 contains two genes, rpf1 and rpf2, encoding proteins with similarities to the essential resuscitation-promoting factor (Rpf) of Micrococcus luteus. Both the Rpf1 (20.4 kDa) and Rpf2 (40.3 kDa) proteins share the so-called Rpf motif, a highly conserved protein domain of approximately 70 amino acids, which is also present in Rpf-like proteins of other gram-positive bacteria with a high G+C content of the chromosomal DNA. Purification of the C. glutamicum Rpf2 protein from concentrated supernatants, SDS-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified modified Rpf2 variants with increased or reduced mobility when compared with the calculated size of Rpf2. A Western blot-based enzyme immunoassay demonstrated glycosylation of the Rpf2 variants with higher molecular masses. Galactose and mannose were identified as two components of the oligosaccharide portion of the Rpf2 glycoprotein by capillary gas chromatography coupled to mass spectrometry. The Rpf2 protein was localized on the surface of C. glutamicum with the use of immuno-fluorescence microscopy. C. glutamicum strains with defined deletions in the rpf1 or rpf2 gene or simultaneous deletions in both rpf genes were constructed, indicating that the rpf genes are neither individually nor collectively essential for C. glutamicum. The C. glutamicum rpf double mutant displayed slower growth and a prolonged lag phase after transfer of long-stored cells into fresh medium. The addition of supernatant from exponentially growing cultures of the rpf double mutant, the wild type or C. glutamicum strains with increased expression of the rpf1 or rpf2 gene significantly reduced the lag phase of long-stored wild-type and rpf single mutant strains, but addition of purified His-tagged Rpf1 or Rpf2 did not. In contrast, the lag phase of the C. glutamicum rpf double mutant was not affected upon addition of these culture supernatants.

Amino Acid Motifs↗

PubMed2004

Construction and validation of cDNA-based Mt6k-RIT macro- and microarrays to explore root endosymbioses in the model legume Medicago truncatula.

To construct macro- and microarray tools suitable for expression profiling in root endosymbioses of the model legume Medicago truncatula, we PCR-amplified a total of 6048 cDNA probes representing genes expressed in uninfected roots, mycorrhizal roots and young root nodules [Nucleic Acids Res. 30 (2002) 5579]. Including additional probes for either tissue-specific or constitutively expressed control genes, 5651 successfully amplified gene-specific probes were used to grid macro- and to spot microarrays designated Mt6k-RIT (M. truncatula 6k root interaction transcriptome). Subsequent to a technical validation of microarray printing, we performed two pilot expression profiling experiments using Cy-labeled targets from Sinorhizobium meliloti-induced root nodules and Glomus intraradices-colonized arbuscular mycorrhizal roots. These targets detected marker genes for nodule and arbuscular mycorrhiza development, amongst them different nodule-specific leghemoglobin and nodulin genes as well as a mycorrhiza-specific phosphate transporter gene. In addition, we identified several dozens of genes that have so far not been reported to be differentially expressed in nodules or arbuscular mycorrhiza thus demonstrating that Mt6k-RIT arrays serve as useful tools for an identification of genes relevant for legume root endosymbioses. A comprehensive profiling of such candidate genes will be very helpful to the development of breeding strategies and for the improvement of cultivation management targeted at increasing legume use in sustainable agricultural systems.

Equipment Design↗

PubMed2004

Comparing expression level-dependent features in codon usage with protein abundance: an analysis of 'predictive proteomics'.

Synonymous codon usage is a commonly used means for estimating gene expression levels of Escherichia coli genes and has also been used for predicting highly expressed genes for a number of prokaryotic genomes. By comparison of expression level-dependent features in codon usage with protein abundance data from two proteome studies of exponentially growing E. coli and Bacillus subtilis cells, we try to evaluate whether the implicit assumption of this approach can be confirmed with experimental data. Log-odds ratio scores are used to model differences in codon usage between highly expressed genes and genomic average. Using these, the strength and significance of expression level-dependent features in codon usage were determined for the genes of the Escherichia coli, Bacillus subtilis and Haemophilus influenzae genomes. The comparison of codon usage features with protein abundance data confirmed a relationship between these to be present, although exceptions to this, possibly related to functional context, were found. For species with expression level-dependent features in their codon usage, the applied methodology could be used to improve in silico simulations of the outcome of two-dimensional gel electrophoretic experiments.

Animals↗

PubMed2004

Comparative genomics identified two conserved DNA modules in a corynebacterial plasmid family present in clinical isolates of the opportunistic human pathogen Corynebacterium jeikeium.

Investigation of 62 clinical isolates of the opportunistic human pathogen Corynebacterium jeikeium revealed that 17 possessed plasmids ranging in size from 7.6 to 14.9 kb. The plasmids formed four groups on DNA restriction analysis. The complete nucleotide sequence of a representative from each group (pK43, pK64, pCJ84, and pB85766) was subsequently determined. Additionally, two plasmids (pCo455 and pCo420) were shown to be derivatives of pK43 and pK64 carrying insertion sequences of the IS3 family. Comparative genomics identified a conserved plasmid backbone consisting of two distinct DNA modules. Conserved motifs in the parAB-repA module indicated that the sequenced plasmids from C. jeikeium are new members of the pNG2 family. Recombinant derivatives of pK43 were shown to replicate in the soil bacterium Corynebacterium glutamicum and in the human pathogen Corynebacterium diphtheriae. The second plasmid module most likely encodes a novel type of DNA invertase. The respective gene is flanked by highly conserved 112-bp inverted repeats. All plasmids are 'loaded' with a characteristic set of genes encoding products of unknown function. Plasmids indistinguishable from pK43 by DNA restriction analysis were identified in different C. jeikeium strains, which revealed 16S-23S rDNA spacer length polymorphisms and specific antibiotic susceptibility profiles, implying a wide dissemination of the plasmid in clinical isolates of C. jeikeium.

Amino Acid Sequence↗

PubMed2003

Qualitative and quantitative proteomics by two-dimensional gel electrophoresis, peptide mass fingerprint and a chemically-coded affinity tag (CCAT).

The chemically-coded affinity tag (CCAT) method combines standard electrophoresis protocols with MALDI-TOF-MS analysis to identify and quantify protein abundances in complex samples in one step. This method is designed to fit into the workflow of SDS-PAGE or two-dimensional electrophoresis (2-DE) only requiring basic proteome laboratory equipment. Prior to electrophoresis two protein samples are separately labelled with a heavy or a light version of the CCAT reagent via reduced cysteines in the proteins. Equal amounts are then combined and electrophoretically separated. Proteins can then be excised from the gel to obtain their peptide mass fingerprint by mass spectrometry. This fingerprint enabled not only identification, but also quantification by comparing relative peak intensities of CCAT-labelled peptides. In this article, we display how the CCAT method can be used to analyse two protein samples in one gel and that the peak intensities of labelled peptides reflect the abundance of a protein in it.

Affinity Labels↗

PubMed2003

Development of a Corynebacterium glutamicum DNA microarray and validation by genome-wide expression profiling during growth with propionate as carbon source.

A DNA microarray was developed to analyse global gene expression of the amino acid-producing bacterium Corynebacterium glutamicum. PCR products representing 93.4% of the predicted C. glutamicum genes were prepared and spotted in quadruplicate onto 3-aminopropyltrimethoxysilane-coated glass slides. The applicability of the C. glutamicum DNA microarray was demonstrated by co-hybridisation with fluorescently labelled cDNA probes. Analysis of the technical variance revealed that C. glutamicum genes detected with different intensities resulting in ratios greater than 1.52 or smaller than -1.52 can be regarded as differentially expressed with a confidence level of greater than 95%. In a validation example, we measured changes of the mRNA levels during growth of C. glutamicum with acetate and propionate as carbon sources. Acetate-grown C. glutamicum cultures were used as reference. At the 95% confidence interval, 117 genes revealed increased transcript levels in the presence of propionate, while 43 genes showed a decreased expression compared with the acetate-grown culture. Global expression profiling confirmed the induction of the prpD2B2C2 gene cluster already known to be essential for propionate degradation via the 2-methylcitrate cycle. Besides many genes of unknown function, the paralogous prpD1B1C1 gene cluster as well as fasI-B (encoding fatty-acid synthase IB), dtsR1 and dtsR2 (components of acyl-CoA carboxylases), gluABCD (glutamate transport system), putP (proline transport system), and pyc (pyruvate carboxylase) showed significantly increased expression levels. Differential expression of these genes was confirmed by real-time reverse transcription (RT) PCR assays.

Carbon↗

PubMed2003

EMMA: a platform for consistent storage and efficient analysis of microarray data.

As a high throughput technique, microarray experiments produce large data sets, consisting of measured data, laboratory protocols, and experimental settings. We have implemented the open source platform EMMA to store and analyze these data. The system provides automated pipelines for data processing and has a modular architecture that can be easily extended. EMMA features detailed reports about spots and their corresponding measurements. In addition to routine data analysis algorithms, the system can be integrated with other components that contain additional data sources (e.g. genome annotation systems).

Algorithms↗

PubMed2003

The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins.

The complete genomic sequence of Corynebacterium glutamicum ATCC 13032, well-known in industry for the production of amino acids, e.g. of L-glutamate and L-lysine was determined. The C. glutamicum genome was found to consist of a single circular chromosome comprising 3282708 base pairs. Several DNA regions of unusual composition were identified that were potentially acquired by horizontal gene transfer, e.g. a segment of DNA from C. diphtheriae and a prophage-containing region. After automated and manual annotation, 3002 protein-coding genes have been identified, and to 2489 of these, functions were assigned by homologies to known proteins. These analyses confirm the taxonomic position of C. glutamicum as related to Mycobacteria and show a broad metabolic diversity as expected for a bacterium living in the soil. As an example for biotechnological application the complete genome sequence was used to reconstruct the metabolic flow of carbon into a number of industrially important products derived from the amino acid L-aspartate.

Amino Acid Sequence↗

PubMed2003

Identification and characterization of the last two unknown genes, dapC and dapF, in the succinylase branch of the L-lysine biosynthesis of Corynebacterium glutamicum.

The inspection of the complete genome sequence of Corynebacterium glutamicum ATCC 13032 led to the identification of dapC and dapF, the last two unknown genes of the succinylase branch of the L-lysine biosynthesis. The deduced DapF protein of C. glutamicum is characterized by a two-domain structure and a conserved diaminopimelate (DAP) epimerase signature. Overexpression of dapF resulted in an 8-fold increase of the specific epimerase activity. A defined deletion in the dapF gene led to a reduced growth of C. glutamicum in a medium with excess carbon but limited ammonium availability. The predicted DapC protein of C. glutamicum shared 29% identical amino acids with DapC from Bordetella pertussis, the only enzymatically characterized N-succinyl-aminoketopimelate aminotransferase. Overexpression of the dapC gene in C. glutamicum resulted in a 9-fold increase of the specific aminotransferase activity. A C. glutamicum mutant with deleted dapC showed normal growth characteristics with excess carbon and limited ammonium. Even a mutation of the two genes dapC and ddh, interrupting both branches of the split pathway, could be established in C. glutamicum. Overexpression of the dapF or the dapC gene in an industrial C. glutamicum strain resulted in an increased L-lysine production, indicating that both genes might be relevant targets for the development of improved production strains.

Amino Acid Isomerases↗

PubMed2003

Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics.

Endogenous plasmids and selectable resistance markers are a fundamental prerequisite for the development of efficient recombinant DNA techniques in industrial microorganisms. In this article, we therefore summarize the current knowledge about endogenous plasmids in amino acid-producing Corynebacterium glutamicum isolates. Screening studies identified a total of 24 different plasmids ranging in size from 2.4 to 95 kb. Although most of the C. glutamicum plasmids were cryptic, four plasmids carried resistance determinants against the antibiotics chloramphenicol, tetracycline, streptomycin-spectinomycin, and sulfonamides. Considerable information is now available on the molecular genetic organization of 12 completely sequenced plasmid genomes from C. glutamicum. The deduced mechanism of plasmid DNA replication and the degree of amino acid sequence similarity among replication initiator proteins was the basis for performing a classification of the plasmids into four distinct C. glutamicum plasmid families.

Base Sequence↗

PubMed2003

Genome-based analysis of biosynthetic aminotransferase genes of Corynebacterium glutamicum.

Due to broad and overlapping substrate specificities, aminotransferases remain the last uncharacterized enzymes from most amino acid biosynthetic pathways in Corynebacterium glutamicum. We report here a complete description of all aminotransferases participating in the biosynthesis of the branched-chain amino acids and phenylalanine in C. glutamicum. We used methods of profile analysis on the newly available genome sequence to systematically search for and characterize members of the four known aminotransferase classes. This led to the discovery of sixteen new, potential aminotransferase encoding genes in the C. glutamicum genome, eleven of which were subsequently characterized experimentally with respect to their participation in different amino acid biosynthetic pathways. Disruption by insertion mutagenesis of ilvE, encoding a branched-chain amino acid aminotransferase, confirmed its function in leucine and isoleucine biosynthesis. Two double mutants lacking both ilvE and genes classified as class I aminotransferases exhibited additional auxotrophic requirements for valine and phenylalanine, respectively. In C. glutamicum the branched-chain amino acid aminotransferase thus participates in four amino acid biosynthetic pathways, for which in case of valine and phenylalanine biosynthesis two additional enzymes with overlapping substrate specificity exist. The novel protein with aminotransferase activity in valine biosynthesis belongs to the very recently described MocR subfamily of GntR-type helix-turn-helix transcriptional regulators, is located upstream of a potential operon of a newly described pyridoxine biosynthetic pathway and when disrupted, gives rise to a pyridoxine auxotrophy. The theoretical and experimental data we present should further provide a solid platform for ongoing research and understanding of the network of aminotransferases which participate in amino acid biosynthesis in C. glutamicum.

Amino Acid Sequence↗

PubMed2003

The putative transcriptional repressor McbR, member of the TetR-family, is involved in the regulation of the metabolic network directing the synthesis of sulfur containing amino acids in Corynebacterium glutamicum.

In order to isolate transcriptional regulatory proteins involved in L-methionine-dependent repression in Corynebacterium glutamicum, proteins binding to the putative promoter region upstream of the metY gene were isolated by DNA affinity chromatography. One of the isolated proteins was identified as a putative transcriptional repressor of the TetR-family by a mass spectrometry fingerprint technique based on the complete C. glutamicum genome sequence. The respective gene, designated mcbR, was deleted in the mutant strain C. glutamicum DR1. Using 2D-PAGE, the protein contents of the C. glutamicum wild type and the mutant strain DR1 grown in media with or without L-methionine supplementation were compared and a set of six proteins was identified. Their abundance was drastically enhanced in the mutant strain and no longer influenced by L-methionine added to the growth medium. The corresponding genes were identified by mass spectrometry fingerprint analysis. They included metY encoding O-acetyl-L-homoserine sulfhydrylase, metK encoding S-adenosyl-methionine synthethase, hom encoding homoserine dehydrogenase, cysK encoding L-cysteine synthase, cysI encoding an NADPH dependant sulfite reductase, and ssuD encoding an alkanesulfonate monooxygenase. Evidently, the putative transcriptional repressor McbR is involved in the regulation of the metabolic network directing the synthesis of L-methionine in C. glutamicum. The C. glutamicum mcbR mutant can be considered to represent a first step in the construction of an L-methionine production strain.

Amino Acid Sequence↗

PubMed2003

Identification and functional analysis of six mycolyltransferase genes of Corynebacterium glutamicum ATCC 13032: the genes cop1, cmt1, and cmt2 can replace each other in the synthesis of trehalose dicorynomycolate, a component of the mycolic acid layer of the cell envelope.

By data mining in the sequence of the Corynebacterium glutamicum ATCC 13032 genome, six putative mycolyltransferase genes were identified that code for proteins with similarity to the N-terminal domain of the mycolic acid transferase PS1 of the related C. glutamicum strain ATCC 17965. The genes identified were designated cop1, cmt1, cmt2, cmt3, cmt4, and cmt5 ( cmt from corynebacterium mycolyl transferases). cop1 encodes a protein of 657 amino acids, which is larger than the proteins encoded by the cmt genes with 365, 341, 483, 483, and 411 amino acids. Using bioinformatics tools, it was shown that all six gene products are equipped with signal peptides and esterase domains. Proteome analyses of the cell envelope of C. glutamicum ATCC 13032 resulted in identification of the proteins Cop1, Cmt1, Cmt2, and Cmt4. All six mycolyltransferase genes were used for mutational analysis. cmt4 could not be mutated and is considered to be essential. cop1 was found to play an additional role in cell shape formation. A triple mutant carrying mutations in cop1, cmt1, and cmt2 aggregated when cultivated in MM1 liquid medium. This mutant was also no longer able to synthesize trehalose di coryno mycolate (TDCM). Since single and double mutants of the genes cop1, cmt1, and cmt2 could form TDCM, it is concluded that the three genes, cop1, cmt1, and cmt2, are involved in TDCM biosynthesis. The presence of the putative esterase domain makes it highly possible that cop1, cmt1, and cmt2 encode enzymes synthesizing TDCM from trehalose monocorynomycolate.

Arabidopsis Proteins↗

PubMed2003

GenDB--an open source genome annotation system for prokaryote genomes.

The flood of sequence data resulting from the large number of current genome projects has increased the need for a flexible, open source genome annotation system, which so far has not existed. To account for the individual needs of different projects, such a system should be modular and easily extensible. We present a genome annotation system for prokaryote genomes, which is well tested and readily adaptable to different tasks. The modular system was developed using an object-oriented approach, and it relies on a relational database backend. Using a well defined application programmers interface (API), the system can be linked easily to other systems. GenDB supports manual as well as automatic annotation strategies. The software currently is in use in more than a dozen microbial genome annotation projects. In addition to its use as a production genome annotation system, it can be employed as a flexible framework for the large-scale evaluation of different annotation strategies. The system is open source.

Amino Acid Sequence↗

PubMed2003

Insights into the genetic organization of the Corynebacterium diphtheriae erythromycin resistance plasmid pNG2 deduced from its complete nucleotide sequence.

The complete nucleotide sequence of the erythromycin resistance plasmid pNG2 from the human pathogen Corynebacterium diphtheriae S601 was determined. The plasmid has a total size of 15,100 bp and contains at least 17 coding regions. Comparative genomics identified conserved motifs within replication initiator proteins of corynebacterial plasmids and a novel nucleotide sequence feature, termed 22-bp box, located downstream of the repA gene. The erythromycin resistance determinant erm(X) is flanked by inverted repeats of the novel insertion sequence IS3504, which may be responsible for a spontaneous deletion of the antibiotic resistance gene region. Furthermore, pNG2 encodes a putative conjugative relaxase, a membrane protein of the natural resistance-associated macrophage protein (Nramp) family and a protein with Nudix hydrolase signature. Expression of the predicted coding regions of pNG2 in Escherichia coli JM109 was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) assays. The detailed annotation of the entire pNG2 sequence provided genetic information regarding its molecular evolution and its role in dissemination of antibiotic resistance genes by horizontal gene transfer.

Amino Acid Sequence↗

PubMed2002

The alanine racemase gene alr is an alternative to antibiotic resistance genes in cloning systems for industrial Corynebacterium glutamicum strains.

The potential of the alanine racemase gene alr from Corynebacterium glutamicum ATCC 13032 to substitute for antibiotic resistance determinants in cloning systems has been investigated. The alr gene was identified by a PCR technique and its nucleotide sequence was determined. The deduced protein revealed the highest amino acid sequence similarity to the Alr protein from Mycobacterium smegmatis with 45% identical and 58% similar amino acids. A defined alr deletion mutant of C. glutamicum displayed a strict dependence on the presence of D-alanine for growth on complex and minimal medium. The alr gene was placed on a novel C. glutamicum vector which is completely free of antibiotic resistance genes. In vivo complementation of the chromosomal alr deletion with alr-carrying vectors permitted growth of the mutant strain in the absence of external D-alanine and provided strong selective pressure to maintain the plasmid. The alr gene enabled the selection of C. glutamicum transformants with a similar efficiency as the tetracycline resistance gene tetA(33). These data provided experimental evidence that the alr gene can be applied as an alternative selection marker to antibiotic resistance genes in industrial C. glutamicum strains. In an application example, the novel deltaalr host-alr(+) vector-system for C. glutamicum was used to overproduce the vitamin D-pantothenic acid.

Alanine Racemase↗

PubMed2002

Strategy to sequence the genome of Corynebacterium glutamicum ATCC 13032: use of a cosmid and a bacterial artificial chromosome library.

The initial strategy of the Corynebacterium glutamicum genome project was to sequence overlapping inserts of an ordered cosmid library. High-density colony grids of approximately 28 genome equivalents were used for the identification of overlapping clones by Southern hybridization. Altogether 18 contiguous genomic segments comprising 95 overlapping cosmids were assembled. Systematic shotgun sequencing of the assembled cosmid set revealed that only 2.84 Mb (86.6%) of the C. glutamicum genome were represented by the cosmid library. To obtain a complete genome coverage, a bacterial artificial chromosome (BAC) library of the C. glutamicum chromosome was constructed in pBeloBAC11 and used for genome mapping. The BAC library consists of 3168 BACs and represents a theoretical 63-fold coverage of the C. glutamicum genome (3.28 Mb). Southern screening of 2304 BAC clones with PCR-amplified chromosomal markers and subsequent insert terminal sequencing allowed the identification of 119 BACs covering the entire chromosome of C. glutamicum. The minimal set representing a 100% genome coverage contains 44 unique BAC clones with an average overlap of 22 kb. A total of 21 BACs represented linking clones between previously sequenced cosmid contigs and provided a valuable tool for completing the genome sequence of C. glutamicum.

Chromosomes, Artificial, Bacterial↗