Host controlled modification in E.coli

Host controlled modification in E.coli.


Host controlled modification is a process many eukaryotic cells undergo and it serves as a method of protection against viral infection.

Restriction, the cleavage of double stranded DNA, occurs if the DNA is not ‘marked' by a methyl group. The host ‘marks' its own DNA to protect it from cleavage, but sometimes the host can ‘mark' the foreign DNA. This leads to an infection and plaque formation.

Bacteriophage λ was used to demonstrate this system using Escherichia coli K-12 and its EcoK system (type I) and Escherichia coli K-12 NTP14 with its EcoR1 system encoded by the plasmid NTP14 (type II).

Two cycles were carried out; the first cycle showed that λ.K. had a efficiency of plating.


Arber and Dussoix first described restriction and modification by the host in 1962. They used bacteriophage λ to show that the phage genetic material is strain-specifically ‘marked' and when transferred to a different strain, no infection was established. This was due to restriction of phage DNA by the host (Cited in Arber & Linn, 1969, p.468).

It is generally considered that host controlled modification acts to preserve integrity and protect against infection.

The aim of this study was to demonstrate host controlled modification using phage λ.K, which had already been propagated on E.coli K-12. The same strain carrying plasmid NTP 14 was also used.

There are three types of host controlled modification systems, of which two will be carefully considered in this report.

Host controlled modification system I

Type I is the most complex system and has been identified in enterobacteria such as Escherichia coli (E.coli). There are three genes coding for this system: hsdS, hsdM and hsdR. The gene hsdR is about 1000 amino acids long and has its own promoter whereas an operon controls the transcription of hsdM and hsdS, which are both 450 - 600 amino acids in length (see figure 1). The way in which the E.coli K-12 (EcoK) genes are transcribed means that it falls into a sub-category of type Ia modification.

The finished product is a multifunctional enzyme consisting of two R polypeptides, two M polypeptides and one S polypeptide. This enzyme has two roles, it is an endonuclease and it acts as a methytransferase. (Bickle & Kruger, 1993)

Endonuclease is an enzyme that cleaves double stranded DNA. The endonuclease is specific to the recognition sequence but in type I systems acts on a different section of DNA, which means that bands cannot be visualised by gel electrophoresis (see figure 2). Endonuclease binds to the specific recognition sequence and if the recognition sequence is methylated (or hemimethylated) then the enzyme disassociates from the DNA. If the sequence is not methylated then a complex is formed and cleavage occurs. ATP is essential for this process. (Wilson & Murray, 1991)

Methyltransferase is a catalyst for the addition of a methyl group to a nucleotide within the recognition sequence. In the case of EcoK, this sequence is AAC(N6)GTGC (see figure 2). The recognition sequence in type I systems is asymmetrical and bipartite; there is a space where any six nucleotides could exist (N6). S-Adenosyl methionine (AdoMet) is essential for methylation as it is the methyl group source. Only the bases cytosine and adenosine can be methylated and in EcoK, the methyl groups are added as shown in figure 2. Type Ia modification systems are the only known methyltransferases to show preference for DNA which is hemimethylated. (Wilson & Murray, 1991)

Host controlled modification system II

The plasmid NTP14is a non-conjugative plasmid carrying Ap resistance, colicin E1 production and the EcoR1 restriction and modification system (O'Connor & Humphreys., 1982). EcoR1 is a type II restriction and modification system, which is the simplest and most common type.

As with type I, the type II system consists of endonuclease and methyltransferase enzymes but they work independently rather than in a complex as seen in type I. The endonuclease gene is 157-576 amino acids in length and the methyltransferase is 228-587 amino acids long. Endonuclease works as a heterodimer and cleaves both DNA strands simultaneously whereas methyltransferase works as a monomer. The recognition sequence for EcoR1 is a palindrome, which is economical, as one protein will react with both strands in any orientation. The cleavage site is located within the sequence (figure 3) so can be visualised by gel electrophoresis. When cleaved, the DNA is left with sticky 5' overhangs. (Wilson & Murray,. 1991)

Efficiency of plating

EOP is a method used to test the susceptibility of an organism to viral infections by measuring plaque formations. Using a non-restricting organism, the EOP would be one, but for organisms carrying restriction genes the EOP is usually 10-3 - 10-5. (Wilson & Murray, 1991)

Results (For raw data and calculations, please see appendix1.)

Cycle one

Cycle 1 consisted of λ.K. plated on both E.coli K-12 and E.coli K-12 NTP14.

More plaques would be expected to grow on E.coli K-12 than on E.coli K-12 NTP14. It was assumed the EOP would be 1 for E.coli K-12 and expected to be somewhere in the region of 10-3 - 10-5 on E.coli K-12 NTP14 (Wilson & Murray, 1991).

Results obtained from this study show that the plaque forming units per ml for E.coli K-12 and E.coli K-12 NTP14 were 6.95x107 pfu/ml and 2.4x104 pfu/ml respectively.

The results from plates 1, 2 and 8 were used as others had to little or too much growth to count; the accepted levels were between 30-300 plaques.

The EOP of λ.K on E.coli K-12 NT14 was calculated to be 3.45 x 10-4. This is within the expected guide as stated by Wilson and Murray (1991).

These results indicate that when grown on E.coli K-12 NTP14, the phage DNA was restricted. This is because the phage DNA had already been ‘marked' by E.coli K-12 using the EcoK type Ia system of modification. The plasmid NTP14 carries a second system of restriction and modification, EcoR1, which is categorised as type II. The mark is strain specific so the second system does not recognise it and undergoes restriction, disabling the phage DNA so no infection occurs.

Discussion /Conclusion


Arber,W., & Linn, S., 1969. DNA Modification and Restriction.Annual reviewof biochemistry, 38, 467-500.

Bickle, T.A., & Kruger, D.H., 1993. Biology of DNA Restriction. Microbiological reviews, 57(2), 434-450.

O'Connor,C.D., & Humphreys, G.O., 1982. Expression of the EcoRI restriction-modification system and the construction of positive-selection cloning vectors. Gene, 20(2), 219-229.

Wilson, G.G., & Murray, N.E., 1991. Restriction and Modification Systems. Annual review of genetics, 25, 585-627.


Appendix 1 - Results.

Cycle 1-

Plated on K12-

0.1ml of 10-5 phage was used.


Number of plaques

Average number of plaques






The average number of plaques per 0.1ml of 10-5 = 69.5

The average number of plaques per 0.1ml of neat = 6.95 x 106

The average number of plaques per ml of neat = 6.95 x 107 pfu/ml

Plated on K12 NTP14-

0.1ml of 10-1 phage was used.


Number of plaques



The average number of plaques per 0.1ml of 10-1 = 240

The average number of plaques per 0.1ml of neat = 2.4 x 103

The average number of plaques per ml of neat = 2.4 x 104 pfu/ml


λ.K on E.coli K-12 NTP14 using- pfu/ml NTP14

pfu/ml K-12


69500000 EOP= 3.45 x 10-4

Cycle 2 -


Phage Plated

Cells Plated

Plaques Formed

Plaques/neat suspension



0.2ml λ.K

0.2ml K-12 NTP14





0.2ml λ.K

0.2ml K-12 NTP14





0.05ml λ.K 1/200

0.2ml K-12



1.272 x 106


0.05ml λ.K 1/200

0.2ml K-12



9.08 x 105


0.05ml λ.K NTP14 1/200

0.2ml K-12



1.324 x 106


0.05ml λ.K NTP14 1/200

0.2ml K-12



1.774 x 106


0.05ml λ.K NTP14 1/200

0.2ml K-12 NTP14



1.248 x 106


0.05ml λ.K NTP14 1/200

0.2ml K-12 NTP14



1.376 x 106


Plates 1+3: λ.K onto NTP14 + λ.K on K-12

575/ 1.272 x 106 = 4.52044 x 10-4

Plates 2+4: λ.K onto NTP14 + λ.K on K-12

7.5/ 9.08 x 105 = 7.76431 x 10-4

Plates 5+7: λ.K NTP14 onto K12 + λ.K NTP14 on NTP14

1.324 x 106/ 1.248 x 106 = 1.061

Plates 2+4: λ.K NTP14 onto K12 + λ.K NTP14 on NTP14

1.774 x 106/ 1.376 x 106 = 1.268

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