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.

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.

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.

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.

Modulation by adrenaline of electrophysiological membrane parameters and contractility in intact and internally perfused single muscle fibres of the crayfish.

The effect of catecholamines on basic membrane characteristics (including labeled ionic fluxes) and contractile parameters was followed in current clamp and voltage clamp conditions in intact muscle fibres and internally perfused muscle fibre segments respectively of the crayfish Astacus fluviatilis; i.e. in muscle fibres which spike and activate tension on calcium principle. Both adrenaline and noradrenaline (6.10(-6) mol/l) facilitated twitch tension induced by graded membrane responses or strontium all-or-none spikes. No effect of isoprenaline was observed. Adrenaline (6.10(-6)-6.10(-5) mol/l) produced an inotropic effect, which appeared with a latency of 2 min and reached its maximum in 5 min. The rates of activation and relaxation of contraction were increased, whilst the latency and the threshold depolarization were decreased. The changes persisted (several tens of min) after washout of adrenaline, depending on concentration and duration of adrenaline application. The resting potential and the strontium spike (Ca2+ replaced with Sr2+) were not influenced and the graded responses were facilitated by adrenaline (from 36.4 +/- 1 mV to 40.0 +/- 2 mV; RP = 77.2 +/- 0.5 mV). Extracellular Ca2+ ions are required for the inotropic effect of adrenaline to occur. The decrease of electrical and contractile responses in nominal calcium-free solutions or after a blockade of Ca2+ influx by Ni2+ ions (1 mmol/l) was relieved by adrenaline. The persistence of inotropic effect of adrenaline was absent, when the extracellular concentration of Ca2+ ions, [Ca2+]0 was decreased from 13.5 to 3.4 mmol/l or the Ni2+ ions were added. The influx of 89Sr2+ ions was decreased in the presence of Ni2+ ions from 24.2 +/- 4.7 pmol.cm-2.s-1 to 11.0 +/- 2.8 pmol.cm-2.s-1, but restored to 20.4 +/- 5.8 pmol.cm-2.s-1 in the presence of adrenaline (6 mumol/l). Adrenaline itself decreased the influx of 89Sr2+ ions, and prolonged the time constant of efflux both in resting and stimulated fibres. The effect of adrenaline is dependent on mobilization of Ca2+ ions from the sarcoplasmic reticulum. First, the inotropic effect of adrenaline was absent in the presence of procaine (blockator of the Ca release channel of the SR), in spite of the increase of the active membrane response (all-or-none procaine action potential); second, adrenaline accelerated the uptake of Ca ions by SR as evidenced by shortening of the restitution processes after caffeine contractures by adrenaline. Membrane calcium currents are increased by adrenaline as a rule; mainly at lower depolarizations (-50 to -20 mV).(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

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.

Convenience of plasmid R6K higher-copy-number deletion derivative for cloning of larger DNA fragments.

Vector properties of plasmid pNH602, a higher-copy-number deletion mutant of plasmid R6K, were tested by cloning the 6.5 Mg/mol BamHI pSa fragment carrying determinants of resistance to four antibiotics in the unique BamHI site of pNH602. The resulting in vitro constructed recombinant plasmid pNH606 was found to be stable, conjugative, multicopy (20 copies of pNH606 per E. coli chromosome were estimated) and to ensure the increased expression of different genes responsible for the antibiotic resistance. The pSa fragment inserted in the BamHI site of plasmid pNH602 (located in Tn2660) was proved to be transposable to other replicons. Recombinant plasmid pNH606 was analyzed using restriction enzymes BamHI and EcoRI and its physical and genetic map was constructed.

RNA polymerase binding sites on a plasmid R6K derivative with increased copy number.

The specific binding of Escherichia coli RNA polymerase molecules to the DNA of plasmid pNH602, a deletion derivative of R6K having an increased copy number, was detected by electron microscopy. Seven strong RNApol binding sites were found on pNH602 DNA linearized with BamHI or EcoRI restriction endonuclease. All of these specific sites occur in genetically defined regions of the pNH602 molecule. Two of them correspond with the recently reported transcription initiation sites within a region essential for plasmid R6K replication.

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.

An in vivo cointegrate of two plasmids from incompatibility group X.

Formation of a recombinant plasmid designated pNH603 was observed when two plasmids from incompatibility group X, the multicopy plasmid pNH602 (a higher-copy-number deletion derivative of R6K) and the oligocopy plasmid R485, coexisted in a single Escherichia coli cell. According to its size and its restriction endonuclease cleavage pattern, plasmid pNH603 is a true cointegrate of pNH602 and R485. An insertion-sequence-like element coming from plasmid R485 is supposed to mediate the fusion of both replicons. The pNH603 copy number (1-2 per chromosome) indicates that the mechanism of replication of the low-copy-number plasmid is dominant in this cointegrate. No dissociation of pNH603 to parental plasmids was observed even in E. coli K-12 recA+ cells. On the other hand, deletion derivatives of four size classes originate from pNH603 in both recA+ and recA hosts. A miniplasmid designated pNH604, a representative of the most frequent 7 Mg/mol size class, was found, in a low number of copies per host chromosome.

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.

DNA-protein interactions during replication of genetic elements of bacteria.

Specific interactions of DNA with proteins are required for both the replication of deoxyribonucleic acid proper and its regulation. Genetic elements of bacteria, their extrachromosomal elements in particular, represent a suitable model system for studies of these processes at the molecular level. In addition to replication enzymes (DNA polymerases), a series of other protein factors (e.g. topoisomerases, DNA unwinding enzymes, and DNA binding proteins) are involved in the replication of the chromosomal, phage and plasmid DNA. Specific interactions of proteins with DNA are particularly important in the regulation of initiation of DNA synthesis. Association of DNAs with the cell membrane also plays an important role in their replication in bacteria.

New replication mutant pNH602 and its relationship to plasmid pAS3, another deletion derivative of plasmid R6K.

A stable deletion derivative pNH602 was obtained when the recently described higher-copy-number point mutant pNH601 of plasmid R6K was introduced to a minicells-producing strain of Escherichia coli. The size of plasmid pNH602 is 18.8 Mg/mol as determined by electron microscopy. The 7.2 Mg/mol fragment of R6K genome missing in pNH602 carries the Smr-determinant and the region finO and, according to the results of restriction analysis, it includes one EcoRI site. With its radioisotopically determined 33 copies of pNH602 per E. coli K-12 chromosome (npc), representing a 23% increase of the point mutant pNH601 and 150% enhancement of R6K npc, plasmid pNH602 differs from another closely related R6K deletion derivative pAS3 of the same size which exhibits only 20 npc. Both pNH602 and pAS3 plasmids are conjugative.

Isolation and characterization of a higher-copy-number mutant of plasmid R6K.

A stable copy-number mutant (pNH601) of plasmid R6K was isolated by selection for increased resistance to ampicillin determined by this plasmid. The size of the mutant plasmid was found to be unchanged (26 Mg/mol) but it is present in 27 copies of pNH601 per E. coli K-12 chromosome which represents a two-fold increase of R6K copy number value. The following genetic properties of pNH601 are reported and compared with those of R6K: conjugative transfer, fertility inhibition of plasmids belonging to other incompatibility groups, incompatibility with plasmid R485 under both non-selective and selective conditions and the integrative suppression of the dnaA ts mutation. The mutant plasmid pNH601 was found to be different from the original R6K in most of these properties.

A rapid and economic method for estimation of the number of plasmid copies in Escherichia coli cells.

An economic method for a rapid estimation of the number of copies of plasmid R6K delta 1 in E. coli cells using lysis of the cells directly on the agarose gel and electrophoretic separation of radioactively labelled plasmid and chromosomal DNA's is described. The method, particularly useful for screening purposes, permits detection of both the CCC and OC forms of plasmids of molar mass 2-150 Mg/mol and it can be applied to other bacterial systems.

Intracellular location of NR1-plasmid DNA in Escherichia coli minicells.

Intracellular location of plasmid NRI (M = 58 Mg/mol, stringent control of replication 1-2 copies per Escherichia coli chromosomal equivalent) was compared with that of plasmid R6D delta 1 (M = 21 Mg/mol, relaxed control of replication, 10-15 copies per E. coli chromosomal equivalent), both in E. coli minicells. Considerable difference in relative distribution of molecules of these two plasmid DNA's between the cytoplasm and the membrane fraction was found when components of the corresponding minicell lysates were fractionated by sedimentation in a double-linear gradient of caesium chloride and sucrose. Also the differences in relative numbers of NRI DNA and R6K delta 1 DNA molecules stably associated with the membrane of minicells, determined by electron-microscopic examination of the fractions containing plasmid DNA-membrane complexes, was evaluated as statistically significant. The association of NRI DNA molecules with E. coli minicell membrane was found to be a much more frequent event than such association of R6K delta 1 molecules. The absolute amount of plasmid DNA associated with membrane in a single minicell corresponds to one molecule for both NRI and R6K delta 1.

Visualization of membrane-associated R-plasmid DNA in fraction of Escherichia coli minicell lyzate.

Minicells of Escherichia coli P678-54 containing plasmid RIdrd19 were submitted to careful controlled lysis. By sedimentation of the resulting lyzate in a sucrose gradient, the material absorbing at 260 nm was separated into three distinct bands. Among the most rapidly sedimenting particles, double-stranded topological circles of DNA attached to patches of membrane were visualized by electron microscopy, while single-stranded molecules (probably RNA) with associated proteins were detected in the medium band. Covalently closed and open circles of the RIdrd19 DNA were found at the top of the gradient. Their contour lengths correspond to the size of the DNA sedimenting together with the membrane in the first peak. This finding implies a direct intracellular interaction between RIdrd19 DNA and membrane in E. coli minicelle.