Abstract

Efficient electroporation of Escherichia coli with plasmid DNA isolated from Corynebacterium glutamicum depends on the use of Mcr-deficient E. coli strains. The transformation frequency increased nearly 800-fold when the Mcr-deficient E. coli DH5αMCR was used instead of E. coli DH5α. We used E. coli strains with different mutations in the methyl-specific restriction systems to show that McrBC-deficiency is sufficient to generate this effect. The results imply that C. glutamicum DNA contains methylcytosine in specific sequences recognized by the E. coli McrBC system.

REFERENCES

1
Bickle
T.A.
Krüger
D.H.
(
1993
)
Biology of DNA restriction
Microbiol. Rev.
 ,
57
,
434
450
.
2
Waite-Rees
P.A.
Keating
C.J.
Moran
L.S.
Slatko
B.E.
Hornstra
L.J.
Benner
J.S.
(
1991
)
Characterization and expression of the Escherichia coli Mrr restriction system
J. Bacteriol.
 ,
173
,
5207
5219
.
3
Raleigh
E.A.
Wilson
G.
(
1986
)
Escherichia coli K-12 restricts DNA containing 5-methylcytosine
.
83
, In
Proc. Natl. Acad. Sci. USA
, pp
9070
9074
.
4
Raleigh
E.A.
(
1992
)
Organization and function of the mcrBC genes of Escherichia coli K-12
Microbiol.
 ,
6
,
1079
1086
.
5
Martin
J.F.
(
1989
)
Molecular genetics of amino acid-producing corynebacteria
In
Society for General Microbiology Symposium
Baumberg
S.
Hunter
I.
Rhodes
M.
, Eds) Vol.
44
, pp
25
59
.
6
Schäfer
A.
Kalinowski
J.
Simon
R.
Seep-Feldhaus
A.-H.
Pühler
A.
(
1990
)
High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria
J. Bacteriol.
 ,
172
,
1663
1666
.
7
Schwarzer
A.
Pühler
A.
(
1991
)
Manipulation of Corynebacterium glutamicum by gene disruption and replacement
Bio/Technology
 ,
9
,
84
87
.
8
Grant
S.G.N.
Jessee
J.
Bloom
F.R.
Hanahan
D.
(
1990
)
Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methyylation-restriction mutants
.
87
, In
Proc. Natl. Acad. Sci. USA
, pp
4645
4649
.
9
Raleigh
E.A.
Murray
N.E.
Revel
H.
Blumenthal
R.M.
Westaway
D.
Reith
A.D.
Rigby
P.W.J.
Elhai
J.
Hanahan
D.
(
1988
)
McrA and McrB restriction pheno-types of some E. coli strains and implications for gene coning
Nucleic Acids Res.
 ,
16
,
1563
1575
.
10
Gough
J.A.
Murray
N.E.
(
1983
)
Sequence diversity among related genes for recognition of specific targets in DNA molecules
J. Mol. Biol.
 ,
166
,
1
19
.
11
Jerpseth
B.
Greener
A.
Short
J.M.
Viola
J.
Kretz
P.L.
(
1993
)
New restriction-minus derivates of XL1-Blue E. coli cells
Strategies
 ,
6
,
24
.
12
Jäger
W.
Schäfer
A.
Pühler
A.
Labes
G.
Wohlleben
W.
(
1992
)
Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the Gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans
J. Bacteriol.
 ,
174
,
5462
5465
.
13
Dower
W.J.
Miller
J.F.
Ragsdale
C.W.
(
1988
)
High efficiency transformation of E. coli by high voltage electroporation
Nucleic Acids Res.
 ,
16
,
6127
6145
.
14
Santamaria
R.
Gil
J.A.
Mesas
J.M.
Martin
J.F.
(
1984
)
Characterization of an endogenous plasmid and development of cloning vectors and a transformation system in Brevibacterium lactofermentum
J. Gen. Microbiol.
 ,
130
,
2237
2246
.
15
Wang
P.
Harvey
S.S.
Sims
P.F.G.
Broda
P.
(
1992
)
The construction of Streptomyces cyaneus genomic libraries in Escherichia coli is dependent upon the use of Mcr-deficient strains
Gene
 ,
119
,
127
129
.