High efficiency electroporation of intact<i>Corynebacterium glutamicum</i>cells

Liebl, Wolfgang; Bayerl, Anja; Schein, B.; Stillner, U.; Schleifer, Karl‐Heinz

doi:10.1111/j.1574-6968.1989.tb03677.x

Cited by 121 publications

(7 citation statements)

References 8 publications

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“…3). Identical results have been reported by Liebl et al [25] and Bonamy et al [27]. After 4.3 ms cell survival decreased drastically, probably due to excessive heating generated by the electric pulse [26].…”

Section: Effect Of Pulse Lengthsupporting

confidence: 74%

Electrotransformation of whole cells ofBrevibacteriumsp. R312 a nitrile hydratase producing strain: Construction of a cloning vector

Chion¹,

Duran²,

Arnaud³

et al. 1991

FEMS Microbiology Letters

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A rapid and effective method is described for electroporation of Brevibacterium sp. R312, a coryneform strain producing nitrile hydratase and amidase. The transformation efficiency of the method is 10 8 transformants per /xg of plasmid under optimal conditions. Parameters optimised included field strength (11.8 kV cm-~), pulse length (2.4 ms), plasmid DNA concentration (0.25 /zg ml l) and cell density (101° cells ml-1). Surprisingly, the transformation efficiency did not vary with the growth stage, in contrast to results in the literature. A shuttle vector was constructed containing several unique cloning sites downstream of the SP6 RNA polymerase promoter.

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Section: Effect Of Pulse Lengthsupporting

confidence: 74%

Electrotransformation of whole cells ofBrevibacteriumsp. R312 a nitrile hydratase producing strain: Construction of a cloning vector

Chion¹,

Duran²,

Arnaud³

et al. 1991

FEMS Microbiology Letters

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“…Plasmids pTO10, pTO15, and pTO14 were similarly constructed form pTO101, pTO9, and pTO201 (Table I), respectively. Plasmids were introduced into C. glutamicum R163 by electroporation (31). Competent cells were prepared by growing a 500-ml C. glutamicum R163 culture at 30°C (225 rpm) in LB broth to A 600 ϭ 0.2, washing once with 100 ml of 15% glycerol in water, once with 20 ml of the same solution, and resuspending in 2.5 ml of the same solution.…”

Section: Resultsmentioning

confidence: 99%

Hydrocarbon Rulers in UDP-N-acetylglucosamine Acyltransferases

Wyckoff¹,

Lin²,

Cotter³

et al. 1998

Journal of Biological Chemistry

140

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UDP-GlcNAc acyltransferase (LpxA), the first enzyme of lipid A biosynthesis, catalyzes the transfer of an acyl chain activated on acyl carrier protein (ACP) to UDPGlcNAc. LpxAs are very selective for the lengths of their acyl donor substrates. Escherichia coli LpxA prefers R-3-hydroxymyristoyl-ACP to R-3-hydroxydecanoyl-ACP by a factor of ϳ1000, whereas Pseudomonas aeruginosa LpxA prefers the opposite. E. coli G173M LpxA and the reciprocal P. aeruginosa M169G LpxA show reversed substrate selectivity in vitro and in vivo, demonstrating the existence of precise hydrocarbon rulers in LpxAs.

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“…: ϩ49 221 470 6461; Fax: ϩ49 221 470 5091; Email: r.kraemer@uni-koeln.de. competent, strains were grown in LB in the presence of 0.4% isonicotinic acid hydrazide, 2.5% glycine, and 0.1% Tween 80 (19). Cells were washed four times with ice-cold 10% glycerol.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were washed four times with ice-cold 10% glycerol. Transformation was carried out by electroporation (19). Cells were immediately transferred to BHI medium containing 0.5 M sorbitol (19), incubated for 1 h at 30°C in a shaking flask, and plated on BHI medium containing 20 mg/liter kanamycin.…”

Section: Methodsmentioning

confidence: 99%

Osmo-sensing by N- and C-terminal Extensions of the Glycine Betaine Uptake System BetP of Corynebacterium glutamicum

Peter

Burkovski

Krämer

1998

Journal of Biological Chemistry

142

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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.

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High efficiency electroporation of intactCorynebacterium glutamicumcells

Cited by 121 publications

References 8 publications

Electrotransformation of whole cells ofBrevibacteriumsp. R312 a nitrile hydratase producing strain: Construction of a cloning vector

Electrotransformation of whole cells ofBrevibacteriumsp. R312 a nitrile hydratase producing strain: Construction of a cloning vector

Hydrocarbon Rulers in UDP-N-acetylglucosamine Acyltransferases

Osmo-sensing by N- and C-terminal Extensions of the Glycine Betaine Uptake System BetP of Corynebacterium glutamicum

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