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Technical Reference

GENETIC MARKERS IN E. COLI and EndA+ VERSUS EndA–STRAINS
GENETIC MARKERS in E. coli
Name Description Effect of Mutation
ara-14 Mutation in arabinose metabolism Blocks arabinose catabolism.
araD L-ribulose phosphate 4-epimerase mutation; part of an inducible operon araBAD repressed by L-arabinose Blocks arabinose catabolism.
argA N-Acetylglutamate synthase mutation; inhibited by the presence of arginine Arginine required from growth in minimal media.
cycA Mutation of gene product involved in D-alanine, glycine D-serine and D-cycloserine transport, and an L-alanine carrier Mutants cannot use D-alanine as a carbon source.
dam DNA adenine methylase mutation Blocks methylation of adenine residues in the sequence 5'...GmATC...3'.
dapD Succinyl-diaminopimelate aminotransferase Reflects impaired synthesis of succinyl CoA. Mutants need to be supplemented with succinate or lysine + methionine.
dcm DNA cytosine methylase mutation Blocks methylation of cytosine in the sequence 5'...CmCAGG...3' or 5'...CmCTGG...3'.
deoC Deoxyribose-phosphate aldolase mutation ---
deoR Regulatory gene mutation allowing constitutive expression of genes for deoxyribose synthesis Allows efficient propagation of large plasmids.
dut1 Mutation of deoxyuridine triphosphate, which catalyzes the conversion of dUTP to dUMP and PPi Mutants are impaired in conversion of dUTP to dUMP, leading to higher dUPT pools, which can lead to misincorporation of uracil instead of thymidine. Stable incorporation of dUTP needs mutation in ung gene.
endA1 DNA-specific endonuclease I mutation Improves quality of plasmid DNA isolations.
galE Part of the galETK operon that encodes UCP galactose-4-epimerase Mutant is more resistant to bacteriophage P1 infection.
galK Galactokinase mutation Blocks catabolism of galactose.
galT Galactose-1-phosphate uridylyltransferase mutation Blocks catabolism of galactose.
gyrA96 DNA gyrase mutation Confers resistance to nalidixic acid.
hflA150 Protease mutation that leads to stabilization of cII gene products Leads to high frequency of lysogeny by lambda phages (1).
hflB Gene encodes a possible protease component Leads to high frequency of bacteriophage lambda lysogenization.
hsdR (rK-, mK+) Host DNA restriction and methylation system mutation. Restriction minus, modification positive for the E. coli K strain methylation system Allows cloning without cleavage of transformed DNA by endogenous restriction endonucleases. DNA prepared from this strain can be used to transform rK+ E. coli strains.
hsdS20(rB-, mB-) Mutation of specificity determinant for host DNA restriction and methylation system. Restriction minus, modification minus for the E. coli B strain methylation system Allows cloning without cleavage of transformed DNA by endogenous restriction endonucleases. DNA prepared from this strain is unmethylated by the hsdS20 methylases.
lacIq Overproduction of the lac repressor protein Leads to high levels of the lac repressor protein, inhibiting transcription from the lac promoter.
lacY Galactoside permease mutation Blocks lactose utilization.
lacZΔM15 Partial deletion of β-D-galactosidase gene Allows complementation of β-galactosidase activity by α-complementation sequence in pGEM®Z Vectors. Allows blue/white selection for recombinant colonies when plated on X-Gal.
leuB β-isopropylmalate dehydrogenase mutation Requires leucine for growth on minimal media.
Δ(lon) Deletion of lon protease Reduces proteolysis of expressed proteins.
LysS pLysS plasmid is integrated into the host genome Strains carrying this plasmid will be tet resistant and produce T7 lysozyme, a natural inhibitor of T7 RNA polymerase, thus lowering background transcription of sequences under the control of the T7 RNA polymerase promoter (2).
mcrA Mutation in methylcytosine restriction system Blocks restriction of DNA methylated at the sequence 5'...GmCGC...3'.
mcrB Mutation in methylcytosine restriction system Blocks restriction of DNA methylated at the sequence 5'...AGmCT...3'.
metB Cystathionine γ-synthase mutation Requires methionine for growth on minimal media.
metC Cystathionine beta-lyase mutation; involved in methionine biosynthesis Requires methionine for growth on minimal media.
mtl Mutation in mannitol metabolism Blocks catabolism of mannitol.
mutS Methyl-directed mismatch repair mutation Prevents repair of the newly synthesized, unmethylated strand.
ompT Mutation of protease VII, an outer membrane protein Reduces proteolysis of expressed proteins.
P2 P2 bacteriophage lysogen present in host λ phages containing red and gam genes of λ are growth inhibited by P2 lysogens (3).
proA γ-glutamyl phosphate reductase mutation proA/argD mutant will not block proline synthesis but will be repressed by arginine. Mutants excrete proline on minimal media and are resistant to proline analogs. proA/argD/argR triple mutant grows slowly on minimal media + arginine.
proAB Mutations in proline metabolism Requires proline for growth in minimal media.
recA1, recA13 Mutation in recombination Minimizes recombination of introduced DNA with host DNA, increasing stability of inserts. Inserts are more stable in recA1 than recA13 hosts.
recB, recC, recD Exonuclease V mutations. The Rec BCD trimer (exonuclease V) progressively degrades ssDNA and dsDNA in an ATP-dependent manner to form oligonucleotides; implicated in homologous recombination. Reduces general recombination and affects repair of radiation damage. Allows easier propagation of sequences with inverted repeats.
recF Recombination and repair mutation Mutant cannot repair daughter strand gaps (post-replication repair).
relA ppGpp synthetase I mutation Allows RNA synthesis in the absence of protein synthesis. The ribosome-associated ppGpp synthetase I can sense ribosomes with an uncharged tRNA in the A site, a sign of starvation.
rha Use L-rhamnose, a methylpentose Blocks rhamnose catabolism.
rpsL Mutation in subunit S12 of 30S ribosome Confers resistance to streptomycin.
sbcB Exonuclease I mutation Allows general recombination in recBC mutant strains.
strA Mutant alters ribosome protein S12 Confers resistance to streptomycin.
supB, supC, supG, supL, supM, supN, supO Suppressor mutations Suppresses ochre (UAA) and amber (UAG) mutations.
supD, supE, supF Suppressor mutations Suppresses amber (UAG) mutations.
thi-1 Mutation in thiamine metabolism Mutants require thiamine for growth in minimal media.
thr Threonine biosynthesis mutation Mutants are obligate threonine auxotrophs.
thyA Thymidylate synthase; dTTP biosynthesis Mutants are obligate thymidine auxotrophs.
Tn5 Transposon Encodes resistance to kanamycin.
Tn10 Transposon Encodes resistance to tetracycline.
tonA Mutations in outer membrane protein Confers resistance to bacteriophage T1.
traD36 Transfer factor mutation Prevents transfer of F' episome.
trpC Phosphoribosyl anthranilate isomerase mutation; part of tryptophan biosynthesis pathway  
trpR trpR aporepressor; regulates the biosynthesis of tryptophan and its transport  
tsx T6 and colicin K phage receptor; outer membrane protein involved in specific diffusion of nucleosides; transports the antibiotic albicidin Confers resistance to bacteriophage T6 and colicidin K.
ung1 Uracil-DNA N-glycosylase mutation Allows uracil to exist in plasmid DNA.
xyl-5 Mutation in xylose metabolism Blocks catabolism of xylose.
References
  1. Hoyt, A. et al. (1982) Cell 31, 565.
  2. Studier, R.W. (1991) J. Mol. Biol. 219, 37.
  3. Kaiser, K. and Murry, N. (1985) In: DNA Cloning, Vol. 1, Glover, D., ed. IRL Press Ltd, Oxford, U.K.
EndA+ VERSUS EndA– E. coli STRAINS
 
Endonuclease I is a 12kDa periplasmic protein encoded by the gene endA that degrades double-stranded DNA. The E. coli genotype endA1 refers to a mutation in the wildtype endA gene, which produces an inactive form of the nuclease. E. coli strains with this mutation are referred to as EndA negative (EndA–); the wildtype is indicated as EndA+. The table below contains a list of EndA– and EndA+ E. coli strains. High-quality DNA is easily obtained from both EndA+ and EndA– strains, but special precautions must be taken when working with EndA+ strains to ensure the isolation of high-quality DNA (1). These precautions include the use of modified protocol steps (as indicated in the Technical Bulletins for the Wizard® Plus DNA Purification Systems) and the use of a less rich growth medium (e.g., LB). The modified protocol will eliminate most problems associated with these strains. However, the level of endonuclease I produced is strain-dependent, and the modified protocols may not totally exclude endonuclease I from plasmid DNA prepared from very high endonuclease I-producing strains. In general, we recommend the use of EndA– strains of E. coli whenever possible.

Reference

  1. Schoenfeld, T. et al. (1995) DNA purification: Effects of bacterial strains carrying the endA1 genotype on DNA quality isolated with Wizard® Plasmid Purification Systems. Promega Notes 53, 12.
EndA+ Strains of E. coli EndA– Strains of
E. coli
ABLE® C BJ5182
ABLE® K DH1
BL21(DE3) DH10B
BMH 71-18 DH20
CJ236 DH21
C600 DH5α
DH12S™ JM103
ES1301 JM105
HB101 JM106
HMS174 JM107
JM83 JM108
JM101 JM109
JM110 MM294
KW251 Select96™
LE392 SK1590
M1061 SK1592
NM522 (all NM series strains
 are EndA+)		 SK2267
P2392 SRB
PR700 Stbl2™
Q358 Stbl4™
RR1 SURE®
TB1 TOP10
TG1 XLO
TKB1 XL1-Blue
Y1088 (all Y series strains
 are EndA+)		 XL10-Gold®
 
 

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