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A Burkovski

Publications and source records attributed to A Burkovski.

35 records · Page 2Linked to original sources
PubMed1998

Osmo-sensing by N- and C-terminal extensions of the glycine betaine uptake system BetP of Corynebacterium glutamicum.

The major uptake carrier for the compatible solute glycine betaine in Corynebacterium glutamicum is the secondary transport system BetP. It is effectively regulated by the external osmolality both on the level of expression and of activity. BetP carries highly charged domains both at the N and at the C terminus. We investigated the role of these extensions in the regulatory response to hyperosmotic stress. Mutants of the betP gene coding for proteins with truncated N- and C-terminal extensions were expressed in the C. glutamicum betP deletion strain DHP1 and were functionally characterized with respect to regulation of activity. The optimum of activation at 1.3 osmol/kg in wild type was shifted in the recombinant strains to about 2.6 osmol/kg in mutants with deletions in the N-terminal part. Deletions in the C-terminal domain resulted in a complete loss of regulation. The altered response to changes in osmolality led to severe consequences in the cellular adaption to hyperosmotic stress. Whereas in the wild type, the steady state level of glycine betaine accumulation is maintained by activity regulation of the BetP system itself, in the mutant with BetP proteins carrying truncations in the C-terminal domain, the observed steady state betaine accumulation was found to be due to a kinetic balance of unregulated glycine betaine uptake by the modifed BetP and efflux via the mechanosensitive efflux channel for compatible solutes at the same time.

Amino Acids, Diamino↗
PubMed1998

Mapping and identification of Corynebacterium glutamicum proteins by two-dimensional gel electrophoresis and microsequencing.

As a prerequisite for proteome analyses of Corynebacterium glutamicum separation of the cytoplasm and the membrane fraction was optimized and two-dimensional (2-D) gel electrophoresis was established. The resulting 2-D protein maps revealed over 1000 silver-stained protein spots separated by isoelectric point and molecular mass for cytoplasmic proteins and approximately 700 silver-stained spots for proteins of the membrane fraction. Proposing a mean size of 1 kbp per gene the complete C. glutamicum genome of 3 Mbp encodes 3000 different proteins; more than half of these can be located using the maps which are presently available. In this study 10 proteins were identified by N-terminal microsequencing, namely the 35 kDa antigen, antigen 84, ATP synthase subunits alpha, gamma and delta, cysteine synthase, elongation factor G and Ts, enolase, and rotamase. For seven sequences, corresponding proteins could not be identified. Additionally, two proteins were specifically detected by immunoblotting, a corynebacterial porin and the cytoplasmic protein threonine dehydratase. The methods and 2-D maps established in this study will be the basis for comparative studies of protein expression and a detailed proteome analysis of C. glutamicum.

Amino Acid Sequence↗
PubMed1998

Isoleucine uptake in Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene product.

By complementation analysis of an isoleucine-uptake-deficient Escherichia coli strain, it was shown that a 1.6-kb HindIII-StuI fragment of Corynebacterium glutamicum ATCC 13032, located downstream of the aecD gene, encodes an isoleucine uptake system. Sequence analysis revealed that the complementing fragment carried an open reading frame, termed brnQ, that encodes a protein with sequence similarities to branched-chain amino acid carriers of gram-positive and gram-negative bacteria. The brnQ gene specifies a predominantly hydrophobic protein of 426 amino acid residues with a calculated molecular mass of 44.9 kDa. A topology prediction by neural network computer analysis suggests the existence of 12 hydrophobic segments that most probably form transmembrane alpha-helices. A C. glutamicum mutant strain harboring a defined deletion of brnQ in the chromosome showed a considerably lower isoleucine uptake rate of 0.04 nmol min-1 mg (dry mass)-1 as compared to the wild-type strain rate of 1.2 nmol min-1 mg (dry mass)-1. Overexpression of brnQ by means of a tac promotor resulted in an elevated uptake rate for isoleucine of 11.3 nmol min-1 mg (dry mass)-1. Evidently, the brnQ gene encodes the only transport system in C. glutamicum directing isoleucine uptake.

Amino Acid Sequence↗
PubMed1998

Urea uptake and urease activity in Corynebacterium glutamicum.

When Corynebacterium glutamicum is grown with a sufficient nitrogen supply, urea crosses the cytoplasmic membrane by passive diffusion. A permeability coefficient for urea diffusion of 9 x 10(-7) cm s-1 was determined. Under conditions of nitrogen starvation, an energy-dependent urea uptake system was synthesized. Carrier-mediated urea transport was catalyzed by a secondary transport system linked with proton motive force. With a Km for urea of 9 microM, the affinity of this uptake system was much higher than the affinity of urease towards its substrate (Km approximately 55 mM urea). The maximum uptake velocity depended on the expression level and was relatively low [2-3.5 nmol min-1 (mg dry wt.)-1].

Adenosine Triphosphate↗
PubMed1998

Corynebacterium glutamicum is equipped with four secondary carriers for compatible solutes: identification, sequencing, and characterization of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine betaine carrier, EctP.

Gram-positive soil bacterium Corynebacterium glutamicum uses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in C. glutamicum, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A DeltabetP DeltaputP DeltaproP DeltaectP mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (Km, 48 microM) to ectoine (Km, 132 microM). The proP gene could not be expressed from its own promoter in C. glutamicum; however, expression was observed in Escherichia coli. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed ectP codes for a 615-residue transporter. EctP preferred ectoine (Km, 63 microM) to betaine (Km, 333 microM) and proline (Km, 1,200 microM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP.

Amino Acids, Diamino↗
PubMed1997

Isolation of the Corynebacterium glutamicum glnA gene encoding glutamine synthetase I.

The Corynebacterium glutamicum glutamine synthetase I (GSI) structural gene glnA was cloned by a PCR approach using oligonucleotide primers derived from conserved amino acid sequences of the GSI proteins from various bacteria. Disruption or deletion of this gene in C. glutamicum led to a glutamine auxotrophic phenotype and complete loss of glutamine synthetase activity, indicating the key role of this enzyme in nitrogen metabolism. Additionally, indications for a second glutamine synthetase, GSII, were found.

Amino Acids↗
PubMed1997

Isolation of the putP gene of Corynebacterium glutamicum and characterization of a low-affinity uptake system for compatible solutes.

Corynebacterium glutamicum accumulates the compatible solutes proline, glycine betaine, and ectoine under conditions of high osmolality. Uptake of proline is mediated by both a high-affinity and a low-affinity secondary transport system. The low-affinity uptake system also accepts glycine betaine and ectoine as substrates. In the present study, the gene encoding the high-affinity proline uptake system PutP was isolated by heterologous complementation of Escherichia coli mutant strain WG389, which lacks the transport systems BetT, PutP, ProP, and ProU and is unable to synthesize proline and glycine betaine. This gene (putP) encodes a protein of 524 amino acids that shares identity with the proline transport systems PutP of E. coli, Staphylococcus aureus, Salmonella typhimurium, Haemophilus influenzae, and Klebsiella pneumoniae. Functional studies of PutP synthesized in E. coli mutant strain MKH13, which also lacks the transport systems for compatible solutes and is unable to synthesize glycine betaine, revealed that this carrier system is not regulated by the external osmolality on the level of activity. Km values of 7.6 mM for proline and 1.3 mM for sodium as cotransported ion were determined. Deletion of the putP gene allowed the functional characterization of another proline uptake system with low affinity.

Amino Acid Sequence↗
PubMed1997

Ultrastructure of the Corynebacterium glutamicum cell wall.

The cell wall structure of the Gram-positive Corynebacterium glutamicum was evaluated by electron microscopy of thin sections after freeze-substitution and conventional fixation with glutaraldehyde. For the cell wall an overall thickness of approximately 32 nm was determined, with 8.5 nm corresponding to an outer layer, 6.5 nm to an electron translucent region (ETR) as found in mycobacteria and 17 nm to the peptidoglycan. Knob-like surface structures previously observed in freeze-fracture experiments were detected when cells were conventionally processed with a fixation using glutaraldehyde. By mild treatment with detergents approximately 20 proteins were extracted from the cell wall. From seven of these N-terminal amino acid sequences were determined.

Amino Acid Sequence↗
PubMed1996

Functional and genetic characterization of the (methyl)ammonium uptake carrier of Corynebacterium glutamicum.

Under nitrogen starvation conditions, Corynebacterium glutamicum was found to take up methylammonium at a rate of 20 +/- 5 nmol.min-1.(mg dry weight)-1. The specific activity of this uptake was 10-fold lower when growing the cells under sufficient nitrogen supply, indicating a tight regulation on the expression level. The methylammonium uptake showed Michaelis-Menten kinetics with an Km of 44 +/- 7 microM and was completely inhibited by the addition of 10 microM ammonium. This finding and the fact that methylammonium was not metabolized by C. glutamicum strongly suggests that the uptake carrier actually represents an ammonium uptake system. Methylammonium uptake was strictly dependent on the membrane potential. From the pH optimum and the accumulation of methylammonium in equilibrium, it could be deduced that only one net charge is transported and, thus, that methylammonium is taken up in its protonated form via an uniport mechanism. The amt gene encoding the (methyl)ammonium uptake system was isolated and characterized. The predicted gene product of amt consists of 452 amino acids (Mr = 47,699) and shows 26-33% identity to ammonium transporter proteins from Saccharomyces cerevisiae and Arabidopsis thaliana. According to the hydrophobicity profile, it is an integral membrane protein containing 10 or 11 membrane-spanning segments.

Amino Acid Sequence↗
PubMed1996

Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine.

Corynebacterium glutamicum accumulates glycine betaine under conditions of high osmolarity. Previous work revealed the existence of a high-affinity glycine betaine permease which is osmotically regulated. In the present study, the corresponding gene was cloned. The betP gene, encoding the glycine betaine uptake carrier, was isolated by heterologous complementation of mutant strain Escherichia coli MKH13. From sequence analysis it is predicted to encode a protein of 595 amino acids. This protein shares 36% identity with the choline transport system BetT and 28% identity with the carnitine transport system CaiT of E. coli, as well as 38% identity with a protein with an unknown function from Haemophilus influenzae. Analysis of hydropathy indicated a common structure for all four transport proteins. After heterologous expression of betP in E. coli MKH13, the measured Km values for glycine betaine and the cotransported Na+ were similar to those found in C. glutamicum, whereas the modulation of activity by osmotic gradients was shifted to lower osmotic values.

Amino Acid Sequence↗
PubMed1995

Functional expression of the glutamate uptake system from Corynebacterium glutamicum in Escherichia coli.

Glutamate uptake in the Gram-positive Corynebacterium glutamicum is mediated via a binding protein-dependent transport system, which is encoded by the gluABCD gene cluster. Cloning of these genes in an expression vector and subsequent transformation of the resulting plasmid allows different strains of the Gram-negative bacterium Escherichia coli to grow on glutamate as sole carbon and nitrogen source. However, overexpression of the glutamate uptake system results in growth inhibitory effects, probably due to the particular topology of the binding protein.

Biological Transport, Active↗
PubMed1995

Glutamate excretion in Escherichia coli: dependency on the relA and spoT genotype.

Glutamate excretion due to amino acid starvation was investigated in "stringent" and "relaxed" strains of Escherichia coli. The observed excretion process is relA-dependent, carrier-mediated, and glutamate-specific. After induction, excretion was detected within less than 2 min and continued for more than 5 h with a rate of 7-10 nmol (mg dry weight)-1 min-1. Using carbonyl cyanide m-chlorophenylhydrazone or polymyxin B nonapeptide, together with valinomycin, it was shown that glutamate excretion is driven by the membrane potential.

Biological Transport↗
PubMed1994

Hybrid Fo complexes of the ATP synthases of spinach chloroplasts and Escherichia coli. Immunoprecipitation and mutant analyses.

Hybrid Fo complexes of the ATP synthases of spinach chloroplast (CFo) and Escherichia coli (EFo) were investigated. Immunoprecipitations with polyclonal antibodies against the different Fo subunits clearly revealed that hybrid Fo complexes derived from CFo subunit III and EFo subunits a and b were formed in vivo. In addition, the ATPase activities of the hybrid ATP synthase, measured in everted cytoplasmic membranes of an atpE mutant strain transformed with the atpH gene coding for CFo III, were comparable to activities obtained for the same mutant strain complemented with the atpE gene (EFo c). Nevertheless, CFo III was not able to replace EFo c functionally, since the strain containing the hybrid ATP synthase was not able to grow on succinate. In order to investigate the reason for this lack of function, hybrid proteolipids of CFo III and EFo c were constructed. Only a chimaeric protein comprising the seven N-terminal amino acid residues from CFo III and the remaining part of EFo c was able to replace wild-type EFo c, whereas hybrid proteins with 13 and 33 N-terminal amino acids of CFo III were not functional. The results suggested that a network of interactions between the subunits essential for proton translocation and/or coupling of the F1 part exists, which was optimized for each species during evolution, although the overall structure of FoF1 complexes has been conserved.

Amino Acid Sequence↗
PubMed1994

Complementation of Escherichia coli uncD mutant strains by a chimeric F1-beta subunit constructed from E. coli and spinach chloroplast F1-beta.

ATP-synthesizing F0F1-ATPases are complex enzymes consisting of at least eight different subunits. These subunits are conserved during evolution to a very variable degree ranging in pairwise comparison between, for example, Escherichia coli and spinach chloroplast from 20% to 66% identical residues. It was surprising to find that some of the less well conserved subunits like delta and epsilon could replace their E. coli counterparts, whereas the highly conserved beta subunit, which carries the active site, in the E. coli enzyme could not be substituted by spinach chloroplast beta (Lill et al. (1993) Biochim. Biophys. Acta 1144, 278-284). We constructed a chimeric F1-beta subunit consisting of spinach beta in which the 96 N-terminal amino acids were replaced by the respective residue sequence from E. coli beta. Whereas spinach beta did not complement E. coli uncD mutant strains, the chimeric beta subunit restored growth under conditions of oxidative phosphorylation.

Amino Acid Sequence↗
PubMed1993

Complementation of Escherichia coli unc mutant strains by chloroplast and cyanobacterial F1-ATPase subunits.

The genes encoding the five subunits of the F1 portion of the ATPases from both spinach chloroplasts and the cyanobacterium Synechocystis sp. PCC 6803 were cloned into expression vectors and expressed in Escherichia coli. The recombinant subunits formed inclusion bodies within the cells. Each particular subunit was expressed in the respective unc mutant, each unable to grow on non-fermentable carbon sources. The following subunits restored growth under conditions of oxidative phosphorylation: alpha (both sources, cyanobacterial subunit more than spinach subunit), beta (cyanobacterial subunit only), delta (both spinach and Synechocystis), and epsilon (both sources), whereas no growth was achieved with the gamma subunits from both sources. Despite a high degree of sequence homology the large subunits alpha and beta of spinach and cyanobacterial F1 were not as effective in the substitution of their E. coli counterparts. On the other hand, the two smallest subunits of the E. coli ATPase could be more effectively replaced by their cyanobacterial or chloroplast counterparts, although the sequence identity or even similarity is very low. We attribute these findings to the different roles of these subunits in F1: The large alpha and beta subunits contribute to the catalytic centers of the enzyme, a function rendering them very sensitive to even minor changes. For the smaller delta and epsilon subunits it was sufficient to maintain a certain tertiary structure during evolution, with little emphasis on the conservation of particular amino acids.

Amino Acid Sequence↗