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Andreas Burkovski

Publications and source records attributed to Andreas Burkovski.

At least 19 recordsLinked to original sources
PubMed2007

Sugar transport systems of Bifidobacterium longum NCC2705.

Here we present the complement of the carbohydrate uptake systems of the strictly anaerobic probiotic Bifidobacterium longum NCC2705. The genome analysis of this bacterium predicts that it has 19 permeases for the uptake of diverse carbohydrates. The majority belongs to the ATP-binding cassette transporter family with 13 systems identified. Among them are permeases for lactose, maltose, raffinose, and fructooligosaccharides, a commonly used prebiotic additive. We found genes that encode a complete phosphotransferase system (PTS) and genes for three permeases of the major facilitator superfamily. These systems could serve for the import of glucose, galactose, lactose, and sucrose. Growth analysis of NCC2705 cells combined with biochemical characterization and microarray data showed that the predicted substrates are consumed and that the corresponding transport and catabolic genes are expressed. Biochemical analysis of the PTS, in which proteins are central in regulation of carbon metabolism in many bacteria, revealed that B. longum has a glucose-specific PTS, while two other species (Bifidobacterium lactis and Bifidobacterium bifidum) have a fructose-6-phosphate-forming fructose-PTS instead. It became obvious that most carbohydrate systems are closely related to those from other actinomycetes, with a few exceptions. We hope that this report on B. longum carbohydrate transporter systems will serve as a guide for further in-depth analyses on the nutritional lifestyle of this beneficial bacterium.

Bifidobacterium↗
PubMed2007

A glucose kinase from Mycobacterium smegmatis.

Carbon metabolism and regulation is poorly understood in mycobacteria, a genus that includes some major pathogenic species like Mycobacterium tuberculosis and Mycobacterium leprae. Here, we report the identification of a glucose kinase from Mycobacterium smegmatis. This enzyme serves in glucose metabolism and global carbon catabolite repression in the related actinomycete Streptomyces coelicolor. The gene, msmeg1356 (glkA), was found by means of in silico screening. It was shown that it occurs in the same genetic context in all so far sequenced mycobacterial species, where it is located in a putative tricistronic operon together with a glycosyl hydrolase and a putative malonyl-CoA transacylase. Heterologous expression of glkA in an Escherichia coli glucose kinase mutant led to the restoration of glucose growth, which provided in vivo evidence for glucose kinase function. GlkA(Msm) was subsequently overproduced in order to study its enzymatic features. We found that it can form a dimer and that it efficiently phosphorylates glucose at the expense of ATP. The affinity constant for glucose was with 9 mM about eight times higher and the velocity was about tenfold slower when compared to the parallel measured glucose kinase of S. coelicolor. Both enzymes showed similar substrate specificity, which consists in an ATP-dependent phosphorylation of glucose and no, or very inefficient, phosphorylation of the glucose analogues 2-deoxyglucose and methyl alpha-glucoside. Hence, our data provide a basis for studying the role of mycobacterial glucose kinase in vivo to unravel possible catalytic and regulatory functions.

Adenosine Triphosphate↗
PubMed2007

Nitrogen metabolism and nitrogen control in corynebacteria: variations of a common theme.

The published genome sequences of Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium glutamicum and Corynebacterium jeikeium were screened for genes encoding central components of nitrogen source uptake, nitrogen assimilation and nitrogen control systems. Interestingly, the soil-living species C. efficiens and C. glutamicum exhibit a broader spectrum of genes for nitrogen transport and metabolism than the pathogenic species C. diphtheriae and C. jeikeium. The latter are characterized by gene decay and loss of functions like urea metabolism and nitrogen-dependent transcription control. The global regulator of nitrogen regulation AmtR and its DNA-binding motif are conserved in C. diphtheriae, C. efficiens and C. glutamicum, while in C. jeikeium, an AmtR-encoding gene as well as putative AmtR-binding motifs are missing.

Amino Acid Motifs↗
PubMed2006

Mutation-induced metabolite pool alterations in Corynebacterium glutamicum: towards the identification of nitrogen control signals.

The influence of glutamate dehydrogenase activity on nitrogen regulation in Corynebacterium glutamicum was investigated. As shown by RNA hybridization experiments deletion of the gdh gene results in a rearrangement of nitrogen metabolism. Even when sufficiently supplied with nitrogen sources, a gdh deletion strain showed the typical nitrogen starvation response of C. glutamicum. These changes in transcription correlate with distinct alterations of intracellular metabolite pattern. Metabolite analyses of different mutant strains and the wild type indicated that ammonium and 2-oxoglutarate might influence the nitrogen regulation system of C. glutamicum cells.

Corynebacterium glutamicum↗
PubMed2006

Application of global analysis techniques to Corynebacterium glutamicum: new insights into nitrogen regulation.

The regulation of nitrogen metabolism in the amino acid producer Corynebacterium glutamicum was subject of research for several decades. While previous studies focused on single enzymes or pathways, the publication of the C. glutamicum genome sequence gave a fresh impetus to research, since a global investigation of metabolism and regulation networks became possible based on these data. This communication summarizes the advances made by different studies, in which global analysis approaches were used to characterize the C. glutamicum nitrogen starvation response. A combination of bioinformatics approaches, transcriptome and proteome analyses as well as chemostat experiments revealed new insights into the nitrogen control network of C. glutamicum. C. glutamicum reacts to a limited nitrogen supply with a rearrangement of the cellular transport capacity, changes in metabolic pathways for nitrogen assimilation and amino acid biosynthesis, an increased energy generation and increased protein stability. With the aid of chemostat experiments, in which different growth rates were obtained by nitrogen limitation, general starvation effects could be distinguished from specific nitrogen limitation-dependent changes. The core adaptations on the level of transcription are controlled by the master regulator of nitrogen control, the TetR-type protein AmtR. This global regulator governs transcription of at least 33 genes via binding to a palindromic consensus motif (AmtR box). Genes with AmtR box-containing promoters were identified by genome-wide screening and validated, besides by other methods, by transcriptome analyses using DNA microarrays.

Bacterial Proteins↗
PubMed2006

Ammonium toxicity in bacteria.

Although an excellent nitrogen source for most bacteria, ammonium was-in analogy to plant and animal systems-assumed be detrimental to bacteria when present in high concentrations. In this study, we examined the effect of molar ammonium concentrations on different model bacteria, namely, Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. The studied bacteria are highly resistant to ammonium. When growth was impaired upon addition of molar (NH4)2SO4 concentrations, this was not caused by an ammonium-specific effect but was due to an enhanced osmolarity or increased ionic strength of the medium. Therefore, it was concluded that ammonium is not detrimental to C. glutamicum and other bacteria even when present in molar concentrations.

Bacillus subtilis↗
PubMed2006

Protein and proteome phosphorylation stoichiometry analysis by element mass spectrometry.

Protein phosphorylation stoichiometry was assessed by two analytical strategies. Both are based on element mass spectrometry (ICPMS, inductively coupled plasma mass spectrometry) and simultaneous monitoring of (31)P and (34)S. One strategy employs a combination of 1D gel electrophoresis, in-gel digestion, and final microLC-ICPMS analysis (microLC = capillary liquid chromatography). The other strategy uses the combination of 1D gel electrophoresis, protein blotting, and imLA-ICPMS (imLA = imaging laser ablation). The two methods were evaluated with standard phosphoproteins and were applied to the analysis of the cytoplasmatic proteome of bacterial cells (Corynebacterium glutamicum) and eukaryotic cells (Mus musculus). The eukaryotic proteome was found to exhibit a significantly higher phosphorylation degree (approximately 0.8 mol of P/mol of protein) compared to the bacterial proteome (approximately 0.01 mol of P/mol of protein). Both analytical strategies revealed consistent quantitative results, with the microLC-ICPMS approach providing the higher sensitivity. In summary, two ICPMS-based methods for quantitative estimation of the phosphorylation degree of a cellular proteome are presented which access the native proteome state and do not require any type of label introduction or derivatization.

Phosphorylation↗
PubMed2005

DNA microarray analysis of the nitrogen starvation response of Corynebacterium glutamicum.

Nitrogen is an essential component of nearly all of the complex macromolecules in a bacterial cell, e.g. proteins, nucleic acids, and cell wall components. Accordingly, most prokaryotes have developed elaborate control mechanisms to provide an optimal supply of nitrogen for cellular metabolism and to cope with situations of nitrogen limitation. In this communication, a global analysis of the Corynebacterium glutamicum nitrogen starvation response by transcriptional profiling using DNA microarrays is presented. Our results show that C. glutamicum reacts to nitrogen starvation with a rearrangement of the cellular transport capacity, changes in metabolic pathways concerning nitrogen assimilation and amino acid biosynthesis, and a decreased capacity for protein synthesis.

Adaptation, Physiological↗
PubMed2005

Vanillate metabolism in Corynebacterium glutamicum.

Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the mycolic acids-containing actinomycetes, is able to use the lignin degradation products ferulate, vanillate, and protocatechuate as sole carbon sources. The gene cluster responsible for vanillate catabolism was identified and characterized. The vanAB genes encoding vanillate demethylase are organized in an operon together with the vanK gene, coding for a transport system most likely responsible for protocatechuate uptake. While gene disruption mutagenesis revealed that vanillate demethylase is indispensable for ferulate and vanillate utilization, a vanK mutation does not lead to a complete growth arrest but to a decreased growth rate on protocatechuate, indicating that one or more additional protocatechuate transporter(s) are present in C. glutamicum.

Corynebacterium glutamicum↗
PubMed2005

Regulation of AmtR-controlled gene expression in Corynebacterium glutamicum: mechanism and characterization of the AmtR regulon.

AmtR, the master regulator of nitrogen control in Corynebacterium glutamicum, represses transcription of a number of genes during nitrogen surplus. Repression is released by an interaction of AmtR with signal transduction protein GlnK. As shown by pull-down assays and gel retardation experiments, only adenylylated GlnK, which is present in the cells during nitrogen limitation, is able to bind to AmtR. The AmtR regulon was characterized in this study by a combination of bioinformatics, transcriptome and proteome analyses. At least 33 genes are directly controlled by the repressor protein including those encoding transporters and enzymes for ammonium assimilation (amtA, amtB, glnA, gltBD), urea and creatinine metabolism (urtABCDE, ureABCEFGD, crnT, codA), a number of biochemically uncharacterized enzymes and transport systems (NCgl1099, NCgl1100, NCgl 1915-1918) as well as signal transduction proteins (glnD, glnK). For the AmtR regulon, an AmtR box has been defined which comprises the sequence tttCTATN6AtAGat/aA. Furthermore, the transcriptional organization of AmtR-regulated genes and operons was characterized.

Bacterial Proteins↗
PubMed2005

Adaptation of Corynebacterium glutamicum to ammonium limitation: a global analysis using transcriptome and proteome techniques.

Theresponse of Corynebacterium glutamicum to ammonium limitation was studied by transcriptional and proteome profiling of cells grown in a chemostat. Our results show that ammonium-limited growth of C. glutamicum results in a rearrangement of the cellular transport capacity, changes in metabolic pathways for nitrogen assimilation, amino acid biosynthesis, and carbon metabolism, as well as a decreased cell division. Since transcription at different growth rates was studied, it was possible to distinguish specific responses to ammonium limitation and more general, growth rate-dependent alterations in gene expression. The latter include a number of genes encoding ribosomal proteins and genes for F(o)F(1)-ATP synthase subunits.

Adaptation, Physiological↗
PubMed2005

Characterization of methionine export in Corynebacterium glutamicum.

Corynebacterium glutamicum is known for its effective excretion of amino acids under particular metabolic conditions. Concomitant activities of uptake and excretion systems would create an energy-wasting futile cycle; amino acid export systems are therefore tightly regulated. We have used a DNA microarray approach to identify genes for membrane proteins which are overexpressed under conditions of elevated cytoplasmic concentrations of methionine. One of these genes was brnF, coding for the larger subunit of BrnFE, a previously identified two-component isoleucine export system. By deletion, complementation, and overexpression of the brnFE genes in a C. glutamicum strain, in which the two uptake systems for methionine were inactivated, we identified BrnFE as being responsible for methionine export. In the presence of both substrates in the cytoplasm, BrnFE was found to transport isoleucine and methionine at similar rates. The expression of the brnFE gene cluster depends on an Lrp-type transcription factor and was shown to be strongly induced by increasing cytoplasmic methionine concentration. Methionine was a better inducer than isoleucine, indicating that methionine rather than isoleucine might be the native substrate of BrnFE. When the synthesis of BrnFE was blocked by chloramphenicol, fast methionine export was still observed, but only at greatly increased cytoplasmic levels of this amino acid. This indicates the presence of at least one other methionine export system, presumably with low affinity but high capacity. Under conditions where cytoplasmic methionine does not exceed a concentration of 50 mM, BrnFE is the dominant export system for this amino acid.

Bacterial Proteins↗
PubMed2004

Identification and characterization of the main beta-alanine uptake system in Escherichia coli.

In Escherichia coli, beta-alanine is a direct precursor in the biosynthesis of pantothenic acid (vitamin B5). Although a sufficient beta-alanine supply is crucial for biotechnological vitamin B5 production, nothing was known about beta-alanine transport in E. coli until now. The aim of this work was the characterization of beta-alanine transport by E. coli and the identification and overexpression of the corresponding carrier-encoding gene for the rational improvement of pantothenic acid-producing strains. beta-Alanine uptake was found to be an active process catalyzed by the amino acid carrier CycA. The corresponding gene was cloned and overexpressed, resulting in an increase in the uptake rate, compared with the wild type. In all tested strains, this overexpression led to a strong sensitivity to beta-alanine, but not to the other CycA substrates, such as L-alanine, D-alanine, and glycine. This prevented a direct application for the improvement of pantothenic acid-producing strains by an enhanced precursor supply.

Alanine↗
PubMed2004

Utilization of creatinine as an alternative nitrogen source in Corynebacterium glutamicum.

In order to utilize different nitrogen sources and to survive situations of nitrogen limitation, microorganisms have developed several mechanisms to adapt their metabolism to changes in the nitrogen supply. In this communication, the use of creatinine as an alternative nitrogen source in Corynebacterium glutamicum, the identification of a membrane protein involved in creatinine uptake, the transcriptional regulation of the corresponding gene, and expression regulation of the gene encoding the creatinine deaminase are reported. As shown by mutant analyses, RNA hybridization experiments and real-time PCR, the expression of two genes, crnT and codA, is increased in response to nitrogen limitation, and regulation depends on the global nitrogen regulator AmtR. In addition, synthesis of creatinine deaminase during nitrogen starvation was shown by two-dimensional gel electrophoresis and MALDI-TOF-MS followed by peptide mass fingerprint analysis.

Adaptation, Physiological↗
PubMed2004

Regulation of GlnK activity: modification, membrane sequestration and proteolysis as regulatory principles in the network of nitrogen control in Corynebacterium glutamicum.

P(II)-type signal transduction proteins play a central role in nitrogen regulation in many bacteria. In response to the intracellular nitrogen status, these proteins are rendered in their function and interaction with other proteins by modification/demodification events, e.g. by phosphorylation or uridylylation. In this study, we show that GlnK, the only P(II)-type protein in Corynebacterium glutamicum, is adenylylated in response to nitrogen starvation and deadenylylated when the nitrogen supply improves again. Both processes depend on the GlnD protein. As shown by mutant analyses, the modifying activity of this enzyme is located in the N-terminal part of the enzyme, while demodification depends on its C-terminal domain. Besides its modification status, the GlnK protein changes its intracellular localization in response to changes of the cellular nitrogen supply. While it is present in the cytoplasm during nitrogen starvation, the GlnK protein is sequestered to the cytoplasmic membrane in response to an ammonium pulse following a nitrogen starvation period. About 2-5% of the GlnK pool is located at the cytoplasmic membrane after ammonium addition. GlnK binding to the cytoplasmic membrane depends on the ammonium transporter AmtB, which is encoded in the same transcriptional unit as GlnK and GlnD, the amtB-glnK-glnD operon. In contrast, the structurally related methylammonium/ammonium permease AmtA does not bind GlnK. The membrane-bound GlnK protein is stable, most likely to inactivate AmtB-dependent ammonium transport in order to prevent a detrimental futile cycle under post-starvation ammonium-rich conditions, while the majority of GlnK is degraded within 2-4 min. Proteolysis in the transition period from nitrogen starvation to nitrogen-rich growth seems to be specific for GlnK; other proteins of the nitrogen metabolism, such as glutamine synthetase, or proteins unrelated to ammonium assimilation, such as enolase and ATP synthase subunit F(1)beta, are stable under these conditions. Our analyses of different mutant strains have shown that at least three different proteases influence the degradation of GlnK, namely FtsH, the ClpCP and the ClpXP protease complex.

Amino Acid Sequence↗
PubMed2004

Molecular identification of the urea uptake system and transcriptional analysis of urea transporter- and urease-encoding genes in Corynebacterium glutamicum.

The molecular identification of the Corynebacterium glutamicum urea uptake system is described. This ABC-type transporter is encoded by the urtABCDE operon, which is transcribed in response to nitrogen limitation. Expression of the urt genes is regulated by the global nitrogen regulator AmtR, and an amtR deletion strain showed constitutive expression of the urtABCDE genes. The AmtR repressor protein also controls transcription of the urease-encoding ureABCEFGD genes in C. glutamicum. The ure gene cluster forms an operon which is mainly transcribed in response to nitrogen starvation. To confirm the increased synthesis of urease subunits under nitrogen limitation, proteome analyses of cytoplasmic protein extracts from cells grown under nitrogen surplus and nitrogen limitation were carried out, and five of the seven urease subunits were identified.

ATP-Binding Cassette Transporters↗
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↗