Biomedical subjects

Andreas Burkovski

Publications and source records attributed to Andreas Burkovski.

25 records · Page 2Linked to original sources

PubMed2003

I do it my way: Regulation of ammonium uptake and ammonium assimilation 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, recent advances in our knowledge of ammonium uptake, its assimilation, and connected regulatory systems in Corynebacterium glutamicum are discussed with respect to the situation in the bacterial model organisms Escherichia coli and Bacillus subtilis. The regulatory network of nitrogen control in C. glutamicum differs substantially from that in these bacteria, for example, by the presence of AmtR, the unique "master regulator" of nitrogen control, the absence of a NtrB/NtrC two-component signal transduction system, and a different sensing mechanism in C. glutamicum.

Bacillus subtilis↗

PubMed2003

Towards a phosphoproteome map of Corynebacterium glutamicum.

In this study, the phosphoproteome of Corynebacterium glutamicum, an industrially important soil bacterium of the Corynebacterium/Mycobacterium/Nocardia (CMN) group of Gram-positive bacteria, was investigated by two different detection methods: first, by in vivo radio-labeling using [(33)P]-phosphoric acid with subsequent autoradiography and second, by immunostaining with phosphoamino acid-specific monoclonal antibodies. After two-dimensional gel electrophoresis (2-DE), around 60 [(33)P]-labeled protein spots were visualized and around 90 antibody-decorated protein spots detected; 31 of the protein spots were detected with both methods. By peptide mass fingerprinting, 41 different proteins were identified, namely 5-enolpyruvylshikimate 3-phosphate synthase, aconitase, acyl-CoA carboxylase, acyl-CoA synthetase, ATP (synthase alpha- and beta-chain), carbamoyl-phosphate synthase, citrate synthase, cysteine synthase, DnaK, the elongation factors G, P, Ts and Tu, enolase, fructose bisphosphate aldolase, fumarase, Gap dehydrogenase, glutamine synthetase I, glycine hydroxymethyltransferase, GroEL2, GTPase, heat-inducible transcriptional repressor DnaJ2, inorganic pyrophosphatase, isocitrate dehydrogenase, ketol-acid reductoisomerase, lactate dehydrogenase, leucine-tRNA ligase, lipoamide dehydrogenase, methionine synthase, O-acetylhomoserine sulfhydrylase, pyruvate carboxylase, pyruvate kinase, pyruvate oxidase, ribosomal protein S1, RNA polymerase (beta-subunit), succinyl-CoA:CoA transferase, transketolase and UDP-N-acetylmuramoyl-L-alanine ligase, besides a hypothetical 35k protein and a hypothetical glucose kinase. Both detection techniques were used to create a phosphoproteome map. Additionally, the influence of nitrogen deprivation on the phosphoproteome of C. glutamicum was investigated.

Corynebacterium↗

PubMed2003

Ammonium assimilation and nitrogen control in Corynebacterium glutamicum and its relatives: an example for new regulatory mechanisms in actinomycetes.

Nitrogen is an essential component of nearly all complex macromolecules in a bacterial cell, such as 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 review, recent advances in our knowledge of ammonium uptake, its assimilation, and related regulatory systems in Corynebacterium glutamicum, a Gram-positive soil bacterium used for the industrial production of amino acids, are summarized and discussed with respect to the situation in the bacterial model organisms, Escherichia coli and Bacillus subtilis, and in comparison to the situation in other actinomycetes, namely in mycobacteria and streptomycetes. The regulatory network of nitrogen control in C. glutamicum seems to be a patchwork of different elements. It includes proteins similar to the UTase/GlnK pathway of E. coli and expression regulation by a repressor protein as in B. subtilis, but it lacks an NtrB/NtrC two-component signal transduction system. Furthermore, the C. glutamicum regulation network has unique features, such as a new sensing mechanism. Based on its extremely well-investigated central metabolism, well-established molecular biology tools, a public genome sequence and a newly-established proteome project, C. glutamicum seems to be a suitable model organism for other corynebacteria, such as Corynebacterium diphtheriae and Corynebacterium efficiens.

Corynebacterium↗

PubMed2003

Identification of an anion-specific channel in the cell wall of the Gram-positive bacterium Corynebacterium glutamicum.

A cation-selective channel (porin), designated PorA, facilitates the passage of hydrophilic solutes across the cell wall of the mycolic acid-containing actinomycete Corynebacterium glutamicum. Biochemical and electrophysiological investigations of the cell wall of the mutant strain revealed the presence of an alternative channel-forming protein. This porin was purified to homogeneity and studied in lipid bilayer membranes. It forms small anion-selective channels with a diameter of about 1.4 nm and an average single-channel conductance of about 700 pS in 1 M KCl. The PorBCglut channel could be blocked by citrate in a dose-dependent manner. This result was in agreement with growth experiments in citrate as sole carbon source where growth in citrate was impaired as compared with growth in other carbon sources. The PorBCglut protein was partially sequenced and based on the resulting amino acid sequence of the corresponding gene, which was designated as porB, was identified as an unannotated 381 bp long open reading frame (ORF) in the published genome sequence of C. glutamicum ATCC13032. PorBCglut contains 126 amino acids with an N-terminal extension of 27 amino acids. One hundred and thirty-eight base pairs downstream of porB, we found an ORF that codes for a protein with about 30% identity to PorBCglut, which was named PorCCglut. The arrangement of porB and porC on the chromosome suggested that both genes belong to the same cluster. RT-PCR from overlapping regions between genes from wild-type C. glutamicum ATCC 13032 and its ATCC 13032DeltaporA mutant demonstrated that this is the case and that porB and porC are cotranscribed. The gene products PorBCglut and PorCCglut represent obviously other permeability pathways for the transport of hydrophilic compounds through the cell wall of C. glutamicum.

Amino Acid Sequence↗

PubMed2003

PorA represents the major cell wall channel of the Gram-positive bacterium Corynebacterium glutamicum.

The cell wall of the gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes. The channel-forming polypeptide PorA is a 45-amino-acid acidic polypeptide with an excess of four negatively charged amino acids, which is encoded by the 138-bp gene porA. porA was deleted from the chromosome of C.glutamicum wild-type strain ATCC 13032 to obtain mutant ATCC 13032deltaporA. Southern blot analysis demonstrated that porA was deleted. Lipid bilayer experiments revealed that PorA was not present in the cell wall of the mutant strain. Searches within the known chromosome of C. glutamicum by using National Center for Biotechnology Information BLAST and reverse transcription-PCR showed that no other PorA-like protein is encoded on the chromosome or is expressed in the deletion strain. The porA deletion strain exhibited slower growth and longer growth times than the C. glutamicum wild-type strain. Experiments with different antibiotics revealed that the susceptibility of the mutant strain was much lower than that of the wild-type C. glutamicum strain. The results presented here suggest that PorA represents a major hydrophilic pathway through the cell wall and that C. glutamicum contains cell wall channels which are not related to PorA.

Anti-Bacterial Agents↗

PubMed2002

Nitrogen assimilation in Corynebacterium diphtheriae: pathways and regulatory cascades.

Genes encoding proteins for ammonium uptake, assimilation, and the nitrogen regulatory system in Corynebacterium diphtheriae were studied on basis of homology searches using Corynebacterium glutamicum genes as query sequences. Regulation of transcription of these genes in response to nitrogen starvation was analyzed by RNA hybridization experiments and knock-out mutants were generated to verify the function of distinct genes. In this communication, we were able to identify the key components of ammonium assimilation pathways and nitrogen regulation in C. diphtheriae. Moreover, we show in this study that molecular biology methods and vectors developed for C. glutamicum can be applied in C. diphtheriae. The results obtained strengthens the role of C. glutamicum as a model organism for mycolic acids-containing actinomycetes.

Corynebacterium diphtheriae↗

PubMed1998

The role of the Corynebacterium glutamicum rel gene in (p)ppGpp metabolism.

To investigate the metabolism of (p)ppGpp in amino-acid-producing coryneform bacteria, a PCR-based strategy using degenerate consensus oligonucleotides was applied to isolate the rel gene of Corynebacterium glutamicum ATCC 13032. The gene consists of 2283 nucleotides and encodes a protein of 760 amino acids with a molecular mass of 84.4 kDa. The amino acid sequence revealed extensive similarities to the related proteins RelA and SpoT of Escherichia coli, which are known to be involved in (p)ppGpp biosynthesis and degradation. The C. glutamicum rel gene is located downstream of the apt gene encoding an adenine phosphoribosyltransferase, and an ORF with similarities to dciAE, which represents part of a dipeptide transport system in E. coli. A C. glutamicum mutant strain carrying a defined deletion in the rel gene was constructed. This mutant failed to accumulate (p)ppGpp in response to amino acid starvation. When overexpressed in E. coli, the C. glutamicum rel gene was able to reverse growth defects caused by an overexpressed relA gene. It is proposed that the C. glutamicum rel gene encodes a bifunctional enzyme with (p)ppGpp synthetase and (p)ppGpp-degrading activities.

Adenine Phosphoribosyltransferase↗