Transformation of spheroplasts and protoplasts of Corynebacterium glutamicum

Thierbach, Georg; Schwarzer, A.; Pühler, Alfred

doi:10.1007/bf00265819

Cited by 37 publications

(19 citation statements)

References 14 publications

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“…The results presented in this report show that in- (Thierbach et al 1988). With high-voltage electroporation TE is 103 for intact cells compared with 105 for osmotically sensitive cells.…”

Section: Resultsmentioning

confidence: 57%

“…DNA concentration was determined spectrophotometrically. One optical density unit at )~ = 260 nm corresponds to 50 Ixg ml-1 (see Thierbach et al 1988). Plasmid DNA and fragments cleaved by restriction enzymes were analysed by agarose gel electrophoresis according to Maniatis et al (1982).…”

Section: Methodsmentioning

confidence: 99%

“…Osmotically sensitive cells, retaining the original rod shape, were prepared in a growth medium containing 0.5% glycine (Thierbach et al 1988), except that the cells were incubated in buffer with 2.5 mg m1-1 lysozyme and shaked for 3.5 h at 30 ° C.…”

Section: Preparation Of Osmotically Sensitive Cells By Lysozyme Treatmentioning

confidence: 99%

“…Cells were mixed with DNA and filled into the electroporation chamber. After application of a single or of several successive pulses the cells were diluted threefold in Tris/succinate/Mg/ Ca (TSMC) medium supplemented with 0.1% yeast, 0.1% casaminoacids and 0.1% glucose as described by Thierbach et al (1988). After incubation for 1 h-3 h at 30 ° C cells were plated on regeneration medium (1% Agar, 1% tryptone, 0.5% yeast, 1% NaC1, 500 mM sorbitol, 20 mM MgC12, 20 mM CaC12) with 17.5 ~tg kanamycin/ml to select for transformed cells.…”

Section: Electrotransformation Of Cells Frozen Intact and Osmoticallymentioning

confidence: 99%

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Electrotransformation of intact and osmotically sensitive cells of Corynebacterium glutamicum

Wolf

Pühler

Neumann

1989

Appl Microbiol Biotechnol

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Summary. Intact and osmotically sensitive cells ofCorynebacterium glutamicum can be efficiently transformed by electroporation. This was shown by using the plasmid vector pUL-330 (5.2 kb), containing the kanamycin resistance gene of transposon Tn5. The following electric parameters yielded efficient transformation. For intact cells: one exponentially decaying field pulse E = Eo exp ( -t/re) with time constants re = 450-500 ~ts and with initial field intensities of Eo= 35-40 kV cm-l; prepulse temperature 20 ° C. Cell regeneration (survival) was 100%-80%. Transformation efficiency can be increased by an additional freeze and thaw cycle of the cells, prior to electropotation. Lysozyme treated cells (osmotically sensitive) were transformed with three successive pulses of Eo = 25-30 kV cm-1. Cell regeneration under these conditions was found to be 20-30%. The optimum yield of transformants/~tg plasmid-DNA was 3 × 103 for intact cells, 2 × 104 for intact cells which were frozen and thawed twice and 7 x 104 for osmotically sensitive cells if the cell suspension was pulsed at a cell density of 1-3 x 108/ml and at a DNA concentration of 0.2 Ixg/ ml up to _< 2 Ixg/ml. The data obtained for osmotically sensitive cells suggest that the temperature increase accompanying the electric field pulse enhances colony formation and transformation efficiency if the initial prepulse temperature is _> 20 ° C, although regeneration of electroporated C. 91utamicum cells starts to decrease at temperatures > 20 ° C.

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“…The results presented in this report show that in- (Thierbach et al 1988). With high-voltage electroporation TE is 103 for intact cells compared with 105 for osmotically sensitive cells.…”

Section: Resultsmentioning

confidence: 57%

Section: Methodsmentioning

confidence: 99%

Section: Preparation Of Osmotically Sensitive Cells By Lysozyme Treatmentioning

confidence: 99%

Section: Electrotransformation Of Cells Frozen Intact and Osmoticallymentioning

confidence: 99%

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Electrotransformation of intact and osmotically sensitive cells of Corynebacterium glutamicum

Wolf

Pühler

Neumann

1989

Appl Microbiol Biotechnol

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“…We have demonstrated that conjugal transfer of plasmid DNA from gram-negative E. coli to gram-positive coryneform bacteria is possible. Although procedures for transformation of spheroplasts and protoplasts have been developed recently (24), there has been no method available for efficient introduction of foreign plasmid DNA into corynebacteria. This is the first report of high transfer frequencies resulting either from heat treatment of recipient cells before mating or, alternatively, from using a restrictiondeficient mutant of C. glutamicum.…”

mentioning

confidence: 99%

High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria

et al. 1990

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We report on the mobilization of shuttle plasmids from gram-negative Escherichia coli to gram-positive corynebacteria mediated by P-type transfer functions. Introduction of plasmids into corynebacteria was markedly enhanced after heat treatment of the recipient cells. High-frequency plasmid transfer was also observed when the restriction system of the recipient was mutated. On the basis of our data, we conclude that efficient DNA transfer from gram-negative to gram-positive bacteria, at least to coryneform bacteria, is conceivable in certain natural ecosystems.Conjugal transfer of broad-host-range IncP-type resistance plasmids within gram-negative bacterial species is well known (3, 9, 18). Non-self-transmissible plasmids carrying the appropriate origin of transfer (oriT) can be mobilized by IncP plasmids (28). Recent studies have shown that conjugation is a nonspecific process and accounts for most horizontal gene transfer between even phylogenetically remote organisms (7,13,25,26).To investigate the possibility of conjugal plasmid transfer between Escherichia coli and Corynebacterium glutamicum, we took advantage of a mobilization system previously developed for genetic engineering of a wide range of gramnegative bacteria (23). This strategy is based on the oriT and the transfer (Tra) functions of IncP-type broad-host-range plasmid RP4 (10) and consists of E. coli mobilizing strains and derivatives of conventional E. coli vectors (pSUP vectors [22,23]). A series of E. coli-C. glutamicum shuttle plasmids was constructed based on mobilizable E. coli vector pSUP102 (22). The 10.6-kilobase prototype shuttle vector pECM1 resulted from fusion of pSUP102 to C. glutamicum vector pCV35 (Fig. 1).Transfer of pECMl to C. glutamicum. For mating experiments, plasmid pECM1 was introduced by transformation into mobilizing strain E. coli S17-1 (23). E. coli S17-1 carries an RP4 derivative integrated into the chromosome which provides the transfer functions necessary for mobilization. By using this donor, plasmid pECM1 was transferred by conjugation as previously described (23) to a nalidixic acidresistant derivative of E. coli MM294 (14) at frequencies between 10-1 and 10-2 (Table 1) per donor cell. For conjugal transfer of pECM1 to coryneform recipient strains, donor strain S17-1(pECM1) was grown to the late-exponential phase in LB medium (15) containing 50 ,ug of kanamycin per ml. Recipient strains were grown in LB medium to an optical density at 580 nm of 3 to 4. About 7 x 108 donor and 3.5 x 109 recipient cells, corresponding to a ratio of 1:5, were mixed and pelleted by centrifugation at 20°C for a short time. The mating mixture was then carefully suspended in about 500 ,ll of LB medium and spread onto a 0.45-pum-pore-size cellulose acetate filter (Millipore Corp., Bedford, Mass.) placed on a prewarmed LB plate. After 20 h of incubation at 30°C, the cells were washed from the filter with 1 ml of LB medium and mechanical agitation. Transconjugants were * Corresponding author.

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