The genetic and biochemical basis of the transformability of Escherichia coli K12

Oishi, Michio; Cosloy, Sharon D.

doi:10.1016/0006-291x(72)90520-7

Cited by 83 publications

(40 citation statements)

References 4 publications

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“…Chaudhury and Smith (5) found nuclease-deficient mutations in the vicinity of the recBC genes, and it was later shown that the ExoV activity can be eliminated by mutations in recD while leaving the bacteria proficient for recombination (1,4,5). This creates a situation similar to that found for the recBC sbcB sbcC background, also ExoV-and Rec+, which Oishi and Cosloy used for transformation of E. coli with linear DNA (8,9,26). Our results show that strains carrying just the recD mutation can also be transformed by linear DNA.…”

Section: Methodsmentioning

confidence: 93%

“…This is due in part to degradation of the incoming DNA by intracellular exonucleases (13,43). Oishi and Cosloy (8,9,26) showed that E. coli strains lacking exonuclease V (ExoV) by virtue of recB or recC mutations can be transformed by linear DNA if they carry as well the sbcB (and sbcC) mutations that restore recombination proficiency to the recB recC mutant. Similarly, recD mutants also are lacking in exonuclease activity, but unlike the recB recC mutants are robust and recombination proficient without the requirement of additional mutations (5).…”

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confidence: 99%

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Chromosomal transformation of Escherichia coli recD strains with linearized plasmids

1989

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Wild-type Escherichia coil are resistant to genetic transformation by purified linear DNA, probably in part because of exonuclease activity. We demonstrate that E. coli containing a recD mutation could be easily transformed by linearized plasmids containing a selectable marker. The marker was transferred to the chromosome by homologous recombination, whereas plasmid markers not in the region of homology were lost.In contrast to some other bacteria, Escherichia coli is not readily transformable by linear pieces of DNA (34). This is due in part to degradation of the incoming DNA by intracellular exonucleases (13,43). Oishi and Cosloy (8,9,26) showed that E. coli strains lacking exonuclease V (ExoV) by virtue of recB or recC mutations can be transformed by linear DNA if they carry as well the sbcB (and sbcC) mutations that restore recombination proficiency to the recB recC mutant. Similarly, recD mutants also are lacking in exonuclease activity, but unlike the recB recC mutants are robust and recombination proficient without the requirement of additional mutations (5).Jasin and Schimmel (14) and Winans et al. (44) used recB recC sbcB strains to transform with linearized plasmids and homologously replace a chromosomal allele with an allele that had been modified. Yamaguchi and Tomizawa (46) and Gutterson and Koshland (12) used strains defective in polA to transfer genes from plasmids to the chromosome. Strains carrying recB, recC, and sbcB grow poorly and generally carry a mutation in sbcC (22). In addition, strains carrying polA are not robust (15). These considerations make the general use of such strains unattractive.Using the E. coli chemotaxis system genes as a chromosomal target, we show here that recD strains can be transformed with linear DNA. The availability of recD alleles that are mutant by virtue of a mini-Tet insertion facilitates the construction of transformable derivatives of strains of interest by one-step bacteriophage-mediated transduction.MATERIALS AND METHODS Strains, media, and chemicals. The bacterial strains, plasmids, and phages used are listed in Tables 1 and 2. LB medium and Tryptone broth were as described previously (2). The lactose phenotypes of the bacterial strains were determined on agar plates containing 5-bromo-4-chloro-3-indoyl-p-D-galactopyranoside (X-Gal) at 20 ,ug/liter and isopropyl-3-D-thiogalactopyranoside (IPTG) at 1 mM. Methyl esterase assays. The activity of the CheB protein, a methyl esterase whose activity is modulated in response to attractants and repellents, can be measured in vivo by the amount of radioactive methanol released from cells that have been incubated with methyl-labeled radioactive methionine (16,18,19,37,41,42). Steady-state cumulative evolution of methanol was measured by the method of Toews and Adler (41) as described by Kehry et al. (16). Stimulus-dependent esterase activity was measured in the flow apparatus as described by Kehry et al. (6,16,17).Transfer of chromosomal DNA to plasmids. The method for transferring chromosomal DNA to plasmids is ...

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Section: Methodsmentioning

confidence: 93%

mentioning

confidence: 99%

Chromosomal transformation of Escherichia coli recD strains with linearized plasmids

1989

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“…E. coli K-12 strain TN102 is a nalidixic acidresistant derivative of the wild-type strain W3110 (13). E. coli K-12 strain JC7623 recB21 recC22 sbcB15 thr thi leu his pro arg rpsL (18) was used for the construction of mini-R64 plasmid pKK610a. Vector plasmid pHSG398 (28) was used for the construction of dual oriT plasmids.…”

Section: Methodsmentioning

confidence: 99%

Initiation and Termination of DNA Transfer during Conjugation of IncI1 Plasmid R64: Roles of Two Sets of Inverted Repeat Sequences within oriT in Termination of R64 Transfer

Furuya

Komano

2000

J Bacteriol

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Intercellular transfer of plasmid DNA during bacterial conjugation initiates and terminates at a specific origin of transfer, oriT. We have investigated the oriT structure of conjugative plasmid R64 with regard to the initiation and termination of DNA transfer. Using recombinant plasmids containing two tandemly repeated R64 oriT sequences with or without mutations, the subregions required for initiation and termination were determined by examining conjugation-mediated deletion between the repeated oriTs. The oriT subregion required for initiation was found to be identical to the 44-bp oriT core sequence consisting of two units, the conserved nick region sequence and the 17-bp repeat A sequence, that are recognized by R64 relaxosome proteins NikB and NikA, respectively. In contrast, the nick region sequence and two sets of inverted repeat sequences within the 92-bp minimal oriT sequence were required for efficient termination. Mutant repeat A sequences lacking NikA-binding ability were found to be sufficient for termination, suggesting that the inverted repeat structures are involved in the termination process. A duplication of the DNA segment between the repeated oriTs was also found after mobilization of the plasmid carrying initiation-deficient but terminationproficient oriT and initiation-proficient but termination-deficient oriT, suggesting that the 3 terminus of the transferred strand is elongated by rolling-circle-DNA synthesis.Intercellular transfer of plasmid DNA during bacterial conjugation is accomplished by the function of transfer genes borne on each conjugative plasmid (for reviews, see references 4 and 15). All conjugative and mobilizable plasmids, such as R64, F, RP4, and R1162, contain oriT sites as cis elements which function as the origin of transfer of plasmid DNA. At the initiation stage of DNA transfer, a site-and strand-specific nick is introduced into the oriT site with a covalent attachment of the cognate relaxase protein to the 5Ј terminus of the nicked strand. The nicked strand is transferred from donor to recipient cells with the 5Ј terminus leading through a putative channel. In the donor cells, replacement strand DNA synthesis reconstitutes the double-stranded plasmid DNA. After one round of DNA transfer, the relaxase-attached 5Ј terminus of the transferred-strand DNA is religated to its 3Ј terminus to reconstitute the circular structure, and complementary-strand synthesis establishes double-stranded plasmid DNA in the recipient cells. Among these steps, the mechanisms by which initiation and termination of conjugative DNA transfer occur are important issues which remain to be elucidated.Initiation and termination of conjugative DNA transfer at the oriT site were extensively studied using a small mobilizable plasmid, R1162 (RSF1010). R1162 oriT consists of a specific nick site and a 10-bp inverted repeat with one mismatch, which is situated 8 bp from the nick site (3, 27). Three R1162 proteins, MobA, MobB, and MobC, form a protein-DNA complex called the relaxosome at oriT (27). From ...

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“…We do not know why addition of exonuclease I is required for extensive degradation of linear DNA, because the mode of DNA hydrolysis by ATP-dependent DNase has not been clarified completely. However, the intermediates that accumulate during partial degradation of DNA with ATP-dependent DNase under various conditions have tails susceptible to exonuclease I (17; Takagi, Y., unpublished observation), and the transformability of E. coli strain K12 is strongly affected in combination with ATP-dependent DNase and exonuclease 1 (18).…”

Section: Methodsmentioning

confidence: 99%

Isolation of Circular DNA Molecules from Whole Cellular DNA by Use of ATP-Dependent Deoxyribonuclease

Mukai

Matsubara

Takagi

1973

Proc. Natl. Acad. Sci. U.S.A.

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Recently, it was shown that ATP-dependent deoxyribonuclease acts upon linear DNA molecules and single-stranded circular molecules, hydrolyzing them into small, acid-soluble fragments. However, the enzyme does not attack doublestranded circular DNA molecules either of the cc or oc form (4-6). Due to this unique property, the enzyme can be used to isolate both cc and oc molecules from a mixture containing a large amount of linear DNA molecules, such as that originating from chromosomes as a result of shear fragmentation.

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The genetic and biochemical basis of the transformability of Escherichia coli K12

Cited by 83 publications

References 4 publications

Chromosomal transformation of Escherichia coli recD strains with linearized plasmids

Chromosomal transformation of Escherichia coli recD strains with linearized plasmids

Initiation and Termination of DNA Transfer during Conjugation of IncI1 Plasmid R64: Roles of Two Sets of Inverted Repeat Sequences within oriT in Termination of R64 Transfer

Isolation of Circular DNA Molecules from Whole Cellular DNA by Use of ATP-Dependent Deoxyribonuclease

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