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M Pátek

Publications and source records attributed to M Pátek.

At least 19 recordsLinked to original sources
PubMed2003

Host-vector system for phenol-degrading Rhodococcus erythropolis based on Corynebacterium plasmids.

The strain Rhodococcus erythropolis CCM2595, which was shown to degrade phenol, was chosen for genetic studies. To facilitate strain improvement using the methods of gene manipulation, the technique of genetic transfer was introduced and cloning vectors were constructed. Using the plasmid pFAJ2574, an electrotransformation procedure yielding up to 7x10(4) transformants/microg DNA was optimized. Escherichia coli- R. erythropolis shuttle vectors were constructed using the replicons pSR1 and pGA1 from Corynebacterium glutamicum. The small vector pSRK21 (5.8 kb) provides six unique cloning sites and selection of recombinant clones using alpha-complementation of beta-galactosidase in E. coli. This vector, exhibiting high segregational stability under non-selective conditions in R. erythropolis CCM2595, was applied to cloning and efficient expression of the gene coding for green fluorescent protein (gfpuv).

Base Sequence↗
PubMed2002

Atypical location of double-strand origin of replication (nic site) on the plasmid pGA1 from Corynebacterium glutamicum.

The double-strand origin of replication (dso) of the rolling-circle-replicating (RC) plasmid pGA1 from Corynebacterium glutamicum was analyzed using the runoff DNA synthesis assay. The site- and strand-specific breakage of double-stranded plasmid DNA, representing the nic site of dso, was localized precisely within the sequence 5'-CTGG decreases AT-3' in the distal part of the pGA1 rep gene. This location of dso differs from the dso positions found on other RC plasmids and is in agreement with the classification of the plasmid pGA1 into a new group of RC plasmids.

Base Sequence↗
PubMed2001

Identification of a novel gene involved in stable maintenance of plasmid pGA1 from Corynebacterium glutamicum.

The cryptic plasmid pGA1 (4.8 kb) from Corynebacterium glutamicum, replicating in the rolling-circle mode, has been reported to contain four open reading frames longer than 200 bp (ORFA/per, ORFA2, ORFB, ORFC/rep). Here we present another pGA1 gene, ORFE (174 bp), located in the region downstream of the per-ORFA2 gene cluster. The ORFE is transcribed into two RNA species in a direction opposite to that of the per-ORFA2 RNA. Introduction of ORFE in trans into the cells harboring the pGA1 derivatives carrying the main stability determinant, the per gene coding for a product that positively influences the pGA1 copy number and maintenance, increased their segregational stability. Mutation of the putative translational start of the ORFE abolished this observed positive effect in trans. ORFE thus codes for a protein acting as an accessory element involved in stable maintenance of plasmid pGA1 and was hence designated the aes gene (accessory effector of stable maintenance).

Amino Acid Sequence↗
PubMed2001

Expression control and specificity of the basic amino acid exporter LysE of Corynebacterium glutamicum.

LysE of Corynebacterium glutamicum belongs to a large new superfamily of translocators whose members are probably all involved in the export of small solutes. Here, the transcript initiation site of lysE, and its divergently transcribed regulator gene, lysG, are identified. Single-copy transcriptional fusions of lysE with lacZ, and titration experiments, show that LysG is the positive regulator of lysE expression enabling its up to 20-fold induction. This induction requires the presence of a coinducer, which is either intracellular L-lysine, or L-arginine. A competition experiment showed that LysE exports these two basic amino acids at comparable rates of about 0.75 nmol min(-1) (mg dry wt)(-1). Although L-histidine and L-citrulline also act as coinducers of lysE expression, these two amino acids are not exported by LysE. As is evident from the analysis of a lysEG deletion mutant, the physiological role of the lysEG system is to prevent bacteriostasis due to elevated L-lysine or L-arginine concentrations that arise during growth in the presence of peptides or in mutants possessing a deregulated biosynthesis pathway. C. glutamicum has additional export activities other than those of LysE for exporting L-histidine, L-citrulline and L-ornithine.

Amino Acid Transport Systems, Basic↗
PubMed1999

Analysis of the Corynebacterium glutamicum dapA promoter.

Deletion and mutational analysis of the promoter P-dapA from Corynebacterium glutamicum was performed to identify regions and particular nucleotides important for its function. An extended -10 region and a stretch of six T's at positions -55 to -50 were found to be the most important elements in the promoter function. The results of mutational analysis of P-dapA are consistent with the conclusions of statistical computer-aided analysis of 44 C. glutamicum promoter sequences.

Base Sequence↗
PubMed1998

An integron of class 1 is present on the plasmid pCG4 from gram-positive bacterium Corynebacterium glutamicum.

The streptomycin/spectinomycin resistance determinant of the 29-kb plasmid pCG4 from Corynebacterium glutamicum was found to be a part of a typical class 1 integron. The sequence analysis revealed that the integron (designated InCg) identified in this Gram-positive bacterium is almost identical to the integron InC present on the plasmid pSA1700 from the Gram-negative bacterium Pseudomonas aeruginosa. Differences in only two base pairs were found in the 3.8-kb sequence. One base substitution (G-->C) is present in the streptomycin/spectinomycin resistance determinant which is thus identical to the aadA2a gene from the integron In6 of the broad-host-range plasmid pSa. The other one (C-->G) is present in the extended -10 region of the integron promoter involved in expression of the antibiotic resistance gene. It was shown that this novel version of the integron promoter displays five times higher activity in both C. glutamicum and Escherichia coli than the original one.

Bacterial Proteins↗
PubMed1998

Analysis of the leuB gene from Corynebacterium glutamicum.

The leuB gene of Corynebacterium glutamicum was found to be present on a 2.2-kb BamHI-SacI chromosomal fragment which complemented the leuB mutation of Escherichia coli. The activity of 3-isopropylmalate dehydrogenase (EC 1.1.1.85), encoded by the leuB gene, was significantly increased in C. glutamicum cells harbouring a plasmid containing the 2.2-kb fragment. The nucleotide sequence of the C. glutamicum leuB coding region (an open reading frame, ORF, of 1020 bp encoding a polypeptide of 340 amino acids with M(r) of 36 144) was determined. The deduced amino acid sequence of the product of this ORF is highly homologous to those of 3-isopropylmalate dehydrogenases from three species of mycobacteria. The transcriptional start site of the leuB gene was localized 35 bp upstream of its translational start; a functional terminator was detected in the 3' flanking region. Northern hybridization analysis showed that the C. glutamicum leuB gene is transcribed as a single monocistronic RNA (approximately 1.2 kb in size). Activity of the leuB promoter was significantly reduced when leucine was present in the growth medium. This suggests the negative regulation of the leuB expression on the transcriptional level in C. glutamicum cells.

3-Isopropylmalate Dehydrogenase↗
PubMed1997

Plasmid pGA1 from Corynebacterium glutamicum codes for a gene product that positively influences plasmid copy number.

The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheriae). This function was confirmed by deletion mapping of the minimal replicon of pGA1 (1.7 kb) which contains only ORFC. Deletion derivatives of pGA1 devoid of ORFA exhibited significant decreases in the copy number in C. glutamicum cells and displayed segregational instability. Introduction of ORFA in trans into the cells harboring these deletion plasmids dramatically increased their copy number and segregational stability. The ORFA gene product thus positively influences plasmid copy number. This is the first report on such activity associated with a nonintegrating bacterial plasmid. The related plasmids pGA1, pSR1, and pNG2 lacking significant homology with any other plasmid seem to be representatives of a new group of plasmids replicating in the rolling-circle mode.

Amino Acid Sequence↗
PubMed1994

Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the gram-positive methylotroph Brevibacterium methylicum by electrotransformation.

Gram-positive facultative methylotrophic coryneform bacterium Brevibacterium methylicum was efficiently transformed with various plasmids using electroporation of intact cells. In addition to the plasmid vectors pEC71 and pZ6-1 constructed on the basis of cryptic plasmids from coryneform bacteria, broad-host-range plasmids pLS5 (derivative of plasmid pMV158 from Streptococcus agalactiae) and RSF1010 belonging to the incompatibility group IncQ from Gram-negative bacteria were found to be present as autonomous structurally unchanged DNA molecules in B. methylicum transformants. With the exception of pZ6-1, all these plasmids were stably maintained in B. methylicum cells grown under non-selective conditions. When plasmid DNAs isolated from B. methylicum were used, the highest efficiency of transformation (10(5) transformants/micrograms DNA) was achieved.

Biotechnology↗
PubMed1994

Leucine synthesis in Corynebacterium glutamicum: enzyme activities, structure of leuA, and effect of leuA inactivation on lysine synthesis.

Enzymes and genes of the isopropylmalate pathway leading to leucine in Corynebacterium glutamicum were studied, and assays were performed to unravel their connection to lysine oversynthesis. The first enzyme of the pathway is inhibited by leucine (Ki = 0.4 mM), and all three enzyme activities of the isopropylmalate pathway are reduced upon addition of this amino acid to the growth medium. Three different DNA fragments were cloned, each resulting in an oversynthesis of one of the three enzymes. The leuA complementing fragment encoding the isopropylmalate synthase was sequenced. The leuA gene is 1,848 bp in size, encoding a polypeptide with an M(r) of 68,187. Upstream of leuA there is extensive hyphenated dyad symmetry and a putative leader peptide, which are features characteristic of attenuation control. In addition to leuA, the sequenced fragment contains an open reading frame with high coding probability whose disruption did not result in a detectable phenotype. Furthermore, the sequence revealed that this open reading frame separates leuA from lysC, which encodes the aspartate kinase initiating the synthesis of all amino acids of the aspartate family. The leuA gene was inactivated in three lysine-secreting strains by insertional mutagenesis. Fermentations were performed, and a roughly 50% higher lysine yield was obtained when appropriate leucine concentrations limiting for growth of the constructed strains were used.

3-Isopropylmalate Dehydrogenase↗
PubMed1994

Transcriptional analysis and regulatory signals of the hom-thrB cluster of Brevibacterium lactofermentum.

Two genes, hom (encoding homoserine dehydrogenase) and thrB (encoding homoserine kinase), of the threonine biosynthetic pathway are clustered in the chromosome of Brevibacterium lactofermentum in the order 5' hom-thrB 3', separated by only 10 bp. The Brevibacterium thrB gene is expressed in Escherichia coli, in Brevibacterium lactofermentum, and in Corynebacterium glutamicum and complements auxotrophs of all three organisms deficient in homoserine kinase, whereas the Brevibacterium hom gene did not complement two different E. coli auxotrophs lacking homoserine dehydrogenase. However, complementation was obtained when the homoserine dehydrogenase was expressed as a fusion protein in E. coli. Northern (RNA) analysis showed that the hom-thrB cluster is transcribed, giving two different transcripts of 2.5 and 1.1 kb. The 2.5-kb transcript corresponds to the entire cluster hom-thrB (i.e., they form a bicistronic operon), and the short transcript (1.1 kb) originates from the thrB gene. The promoter in front of hom and the hom-internal promoter in front of thrB were subcloned in promoter-probe vectors of E. coli and corynebacteria. The thrB promoter is efficiently recognized both in E. coli and corynebacteria, whereas the hom promoter is functional in corynebacteria but not in E. coli. The transcription start points of both promoters have been identified by primer extension and S1 mapping analysis. The thrB promoter was located in an 87-bp fragment that overlaps with the end of the hom gene. A functional transcriptional terminator located downstream from the cluster was subcloned in terminator-probe vectors.

Amino Acid Sequence↗
PubMed1993

Discovery of biologically active peptides in random libraries: solution-phase testing after staged orthogonal release from resin beads.

To speed drug discovery, we developed an approach for identification of individual peptides with a desired biological activity from a library containing millions of peptides. The approach uses sequential orthogonal release of chemically synthesized peptides from insoluble beads, followed by testing in solution. In this system, each bead within a library of beads has one peptide sequence, but peptide molecules are attached to the bead with three types of chemical linkers, including two linkers cleavable at different pH optima. An uncleavable linker keeps some peptide attached to the bead for sequencing positives from the solution assay. Applicability of this discovery technique was documented by identifying ligands for a monoclonal antibody and for the human platelet fibrinogen receptor, glycoprotein IIb/IIIa.

Amino Acid Sequence↗
PubMed1993

Production of threonine by Brevibacterium flavum containing threonine biosynthesis genes from Escherichia coli.

Genes of the threonine operon of Escherichia coli were used for the construction of a Brevibacterium flavum strain excreting threonine. Using the shuttle vector pCEM300 and a newly constructed shuttle vector pEC71 (7.1 kb, Kmr/Nmr), various plasmids carrying E. coli thr genes were prepared. Mutants resistant to the threonine analog 2-amino-3-hydroxyvaleric acid (AHV) were isolated after the ethyl methanesulfonate treatment of B. flavum carrying these recombinant plasmids. A mutant of B. flavum CCM 351 carrying the cloned genes thrA and thrB accumulated 12 g/L of threonine after 48 h of cultivation.

Brevibacterium↗
PubMed1991

Stabilization of a miniderivative of the broad-host-range IncW plasmid pSa by insertion of plasmid R1 parB region.

The plasmid vector pEM100 (13.5 kb) constructed from pGV1106, a miniderivative of the broad-host-range IncW pSa plasmid, and the pAM330 plasmid of Brevibacterium lactofermentum is not stably maintained in Escherichia coli host cells under nonselective growth conditions. By insertion of a 0.9 kb DNA fragment containing the parB locus (responsible for the maintenance of plasmid R1 in E. coli cells) to plasmid pEM100, plasmid pEM110 was prepared which is maintained in a population of E. coli cells growing without a selection pressure very stably.

Brevibacterium↗
PubMed1990

Plasmid shuttle vector with two insertionally inactivable markers for coryneform bacteria.

A new shuttle vector pCEM500 replicating in Escherichia coli and in Brevibacterium flavum was constructed. It carries two antibiotic resistance determinants (Kmr/Gmr from plasmid pSa of Gram-negative bacteria and Smr/Spr from plasmid pCG4 of Corynebacterium glutamicum) which are efficiently expressed in both hosts and can be inactivated by insertion of DNA fragments into the unique restriction endonuclease sites located within them. This vector was found to be stably maintained in B. flavum and can be used for transfer of the cloned genes into this amino-acid-producing coryneform bacterium.

Brevibacterium↗
PubMed1986

Glutamicin CBII, a bacteriocin-like substance produced by Corynebacterium glutamicum.

Corynebacterium glutamicum CBII, in the stationary phase of growth, was found to produce spontaneously a substance resembling bacteriocins by its bactericidal properties. This substance designated glutamicin CBII was observed to exhibit bactericidal activity against coryneform bacteria (12 species tested) but not against unrelated gram-positive (3) and gram-negative (3) bacteria, while its action on bacteria with no quite known relatedness to the coryneform group (14) was found to be variable. Glutamicin CBII was partially purified by precipitation with ammonium sulphate (70% saturation), selective heat precipitation and gel chromatography on Sephadex G-50. The antibacterial substance diffused through cellophane membrane with an approximate cut-off of 10,000 dalton and its sedimentation coefficient was determined to be 1.1 S by ultracentrifugation. Heating at 100 degrees C for 30 min had no effect on its activity. Glutamicin CBII was proved to be resistant to chloroform, trypsin, chymotrypsin, pronase, and subtilisin. According to its staining behaviour and 1H NMR spectra it probably represents a glycoprotein containing only a minor protein component.

Bacteria↗
PubMed1985

New bacteriophage-like particles in Corynebacterium glutamicum.

Three new phage-like particles (CG1, CG2, and CGK1) were isolated from Corynebacterium glutamicum CBII. Particles CG1 and CG2 are DNA phages with long, noncontractile tails, CGK1 is a killer particle according to electron microscopy. A heat-stable low-molecular-weight bacteriocidal substance affecting various coryneform bacteria was observed to be joined to the killer particle CGK1.

Bacteriocins↗