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Why would I do this?
For one of three reasons:
- You want to cut your plasmid DNA (grown in E. coli) with a restriction endonuclease sensitive to E. coli K-12
methylation patterns (Dam [G meATC] or Dcm [C meC(A/T)GG]). Examples are BclI, TGATCA or ScrFI (CCNGG). For a list of endonucleases sensitive to
these, please click here.
- You want to transform your shuttle plasmid DNA grown in E. coli into a strain of eubacteria or archaea that
restricts DNA with this methylation. Genera reported to restrict methylated DNA include Streptomyces, Acholeplasma, Bacillus, Brevibacterium, Corynebacterium
and Haloferax (1-6).
- You want to study the effects of methylation itself, e.g. on expression or DNA repair (7-12).
What strains can I use for this?
Any otherwise suitable strain that lists dam
and dcm in its genotype.
- For routine use, NEB offers ready to use competent cells, dam-/dcm- competent
E. coli (NEB# C2925H/I). We also
supply the strain ER2925 (#E4109S), free with an order or for the cost of shipping.
- For M13 or when LacI repression is needed ,
JM110 is available from ATCC (Tel: 1(301)881 2600; FAX: 1(301)231 5826; Toll-free orders:
1-800-638-6597; (ATCC Web site). JM110 carries an F'
that bears lacIq DEL(lacZ)M15, and is thus able to grow M13 derivatives and,
repress high-copy lac promoters, and complement a-fragment expressed by plasmids and phagemids
derived from pUC vectors.
- When recombination between repeated
sequences is a problem , GM2929 is an alternative (available from the E. coli
Genetic Stock Center at Yale University; (CGSC Web
site). This strain is ER2925 recF. Strains with recF are as defective in
plasmid-by-plasmid recombination as strains with recA (9). Mutations in recA can't be combined with dam because of DNA repair
problems (13, 14).
- A review of available Dam- and Dcm-
strains can be found in ref. 15.
- Note that all enteric bacteria express
Dam. (So do cyanobacteria and Haemophilus spp refs. 16,
17). Derivatives of E. coli B, such as BL21(DE3), T7 Express (NEB#
C2566H/I) and NEB Express (NEB# C2523H/I),
naturally lack Dcm. Most strains used by molecular biologists are derivatives of K-12, and possess both Dam and Dcm
unless otherwise noted.
What do I need to know when working with dam-/dcm- strains?
- We do not recommend these strains as a host for primary cloning/ligation. The dam mutation can result in
an increased mutation rate in the cell (18, 19). This is because Dam affects mismatch repair and the timing of the initiation of DNA replication.
The dam mutation will also reduce the transformation efficiency of competent cells. DNA should be maintained
in a Dam+ strain unless there is a specific need for DNA free of Dam or Dcm methylation.
- C2925 has a low but noticeable frequency of kanamycin resistant mutations. We have observed a low, variable
number of spontaneous Kan resistance mutants in this strain, and also in another commercially available dam
dcm strain. In both strains, tiny colonies appear on Kan plates at a frequency ranging from 1 in 106 to
1 in 107 cells. These tiny colonies will not grow upon restreaking on a fresh Kan plate. This level
of background resistance should not be a problem for routine transformation of plasmids carrying KanR. The reason
for the strain-dependent appearance of incomplete KanR is not known. Mutations in tufG can yield resistance to low
levels of kanamycin (ref. 20). There are also known interactions between ribosomal mutations leading to streptomycin
resistance (rpsL, mutated in both tested strains) and tufG. Hypothetically, a defect in drug transport could reduce
the effective intracellular kanamycin concentration, enabling low-level resistance mechanisms to be effective. Both
tested strains carry a tsx mutation; this gene encodes a channel protein in E. coli that functions in permeation
of nucleosides across the outer membrane. However, this porin is not known to play a role in aminoglycoside permeation.
Any other hints?
- Good microbiological practice. Always
begin with a single colony when growing the strain to make competent cells. Dam+
revertants can arise (expected revertant frequency in an overnight culture < ~10E-6)
and when they do they will have a selective advantage and will accumulate during serial
mass transfer. One subculture to get the cells to exponential phase is OK.
- Maintaining the dam mutation:
The dam mutation in C2925 is the result of insertion by the CamR transposon Tn9.
Although not essential, it's not a bad idea to maintain selection for the insertion by
keeping the strain on chloramphenicol (15 µg/ml). This does not mean that serial transfers are OK if
you maintain selection, however.
- Spectinomycin amplification of plasmid copy
number: Some plasmids (colE1, pBR322, pACYC184 and 177, but NOT pUC derivatives) can
be obtained in higher yields from C2925, as from any other strain, by using an
"amplification" procedure involving inhibition of protein synthesis (15). Since C2925 is CamR, protein synthesis
is not inhibited by the chloramphenicol called for in the original procedure, so use
spectinomycin instead. DO NOT TRY TO GROW C2925 ON SPECTINOMYCIN. The procedure cited is
based on the repression of plasmid number by the plasmid Rop protein or analogue, and on
the independence of plasmid but not host replication initiation from new protein
synthesis. Basically, the host chromosome stops replicating when protein synthesis is
inhibited, but the plasmid keeps replicating and will continue to replicate to very high
copy if the Rop protein is not present. pUC and other very-high-copy number plasmids are
already missing Rop and will not give improved yields with this procedure.
References:
- Holmes, M. L., Nuttall, S. D., and Dyall-Smith, M. L. (1991): Construction and use of halobacterial shuttle vectors and further
studies on Haloferax DNA gyrase. J. Bacteriol. 173, 3807-3813.
- Macaluso, A., and Mettus, A.-M. (1991): Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid
DNA. J. Bacteriol. 173, 1353-1356.
- MacNeil, D. J. (1988): Characterization of a unique methyl-specific restriction system in Streptomyces avermitilis. J. Bacteriol. 170,
5607-5612.
- Sladek, R. L., Nowak, J. A., and Maniloff, J. (1986): Mycoplasma restriction: identification of a new type of restriction specificity
for DNA containing 5-methylcytosine. J. Bacteriol. 165, 219-225.
- Vertes, A. A., Inui, M., Kobayashi, M., Kurusu, Y., and Yukawa, H. (1993): Presence of mrr- and mcr- like restriction systems
in coryneform bacteria. Res. Microbiol. 144, 181-185.
- Chmuzh, E.V., Kashirina, J.G., Tomilova, J.E., Mezentseva, N.V., Dedkov, V.S., Gonchar, D.A., Abdurashitov, M.A., Degtyarev,
S.K. (2005):A Novel Restriction endonuclease BisI from Bacillus subtilis T30, recognizes a methylated DNA sequence 5'- G(m5C)^NGC-3'. Biotekhnologiya 3:
22-26.
- Marinus, M. G. (1987): DNA methylation in Escherichia coli. Ann. Rev. Genet. 21, 113-131.
- Noyer-Weidner, M., and Trautner, T. A. (1993): Methylation of DNA in prokaryotes. Exs 64, 39-108.
- Lahue, R. S., Au, K. F., and Modrich, P. (1989): DNA mismatch correction in a defined system. Science 245, 160-164.
- Kolodner, R. (1985): J. Bacteriol. 163, 1060.
- Marinus, M.G. (2005): Dr. Jekyll and Mr. Hyde: How the MutSLH Repair System Kills the Cell, pp. 413-429. In Higgins, N.P (ed.) The
Bacterial Chromosome. ASM Press, Washington DC.
- Robertson KD, Jones PA. (1997): Dynamic interrelationships between DNA replication, methylation, and repair. Am J Hum Genet. 61:1220-4.
- Marinus, M. G., and Morris, B. (1975): Biological function for 6-methyladenine residues in the DNA of Escherichia coli K12. J.
Mol. Biol. 85, 309-322.
- McGraw, B., and Marinus, M. G. (1980): Isolation and characterization of Dam+ revertants and suppressor mutations that modify
secondary phenotypes of dam-3 strains of Escherichia coli K-12. Mol. Gen. Genet. 178, 309-315.
- Palmer, B. R., and Marinus, M. G. (1994): The dam and dcm strains of Escherichia coli--a review. Gene 143, 1-12.
- Brooks, J. E., Blumenthal, R. M., and Gingeras, T. R. (1983): The isolation and characterization of the Escherichia coli DNA
adenine methylase (dam ) gene. Nucleic Acids Res. 11, 837-851.
- Barbeyron, T., Kean, K., and Forterre, P. (1984): DNA adenine methylation of GATC sequences appeared recently in the Escherichia
coli lineage. J. Bacteriol. 160, 586-590.
- Glickman, B. W, and Radman, M. (1980) Escherichia coli mutator mutants deficient in methylation-instructed DNA mismatch correction. Proc.
Nat. Acad. Sci USA 77, 1063-1067.
- Marinus, M. G., Carraway, M., Frey, A. Z., Brown, L., and Arraj, A. J. (1983) Insertion mutants in the dam gene of Escherichia
coli K-12 . Mol. Gen. Genet. 192, 288-289.
- Kurland, C. G., Hughes, D. and Ehrenberg, M. Limitations of Translational Accuracy (1996) in Escherichia coli and Salmonella: Cellular
and Molecular Biology. ASM Press, Washington, DC
- Sambrook, J. and Russell, D. (2001) Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor,
N.Y., Volume 1, pp. 1.38-1.41.
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