Chromosomal transformation of Escherichia coli recD strains with linearized plasmids

Abstract

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 ...

In Vivo and in Vitro Function of theEscherichia coli Periplasmic Cysteine Oxidoreductase DsbG

“…This fragment was then ligated with either the 1.4-kbp BsaAI fragment of pACYC184 containing the chloramphenicol resistance gene to generate pPB⌬dsbG::Cm or the 1.3-kbp kanamycin resistance cassette from pUC-4K to generate pPB⌬dsbG::Kan. Plasmid pPB⌬dsbG::Cm was linearized and transformed into the recD Ϫ strain D301 (25), and Cm R , Amp S colonies were selected. Additionally, plasmid pPB⌬dsbG::Kan was digested with MluI and SphI, and the fragment containing the Kan R cassette with the dsbG chromosomal flanking regions was recovered and cloned into pBAD39, a vector containing a conditional IPTG-requiring replicon, an ampicillin resistance gene, and a wild type rpsL gene (Table I).…”

One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

“…Such recBC sbcB mutants have been especially useful for recombining in vitro constructed mutations onto the E. coli chromosome by using linear DNA (14). The discovery that recD mutants are recombinase proficient but lack exonuclease V (15, 16) has led to using singly mutated recD derivatives of E. coli (1) in similar gene disruption experiments.…”

“…A systematic mutational analysis of genes in their normal location can provide significant insight into their function. Although a number of general allele replacement methods (1)(2)(3)(4)(5)(6)(7) can be used to inactivate bacterial chromosomal genes, these all require creating the gene disruption on a suitable plasmid before recombining it onto the chromosome. In contrast, genes can be directly disrupted in Saccharomyces cerevisiae by transformation with PCR fragments encoding a selectable marker and having only 35 nt of flanking DNA homologous to the chromosome (8).…”

Epitope tagging of chromosomal genes in Salmonella

“…In enteric bacteria, similar approaches have been hampered by the instability of linear DNA, rapidly degraded by the nuclease activity (ExoV) of the RecBCD recombinase. Efforts to circumvent this problem have involved the use of mutants or conditions inhibiting ExoV (5)(6)(7)(8)(9). However, these systems are not efficient enough to promote recombination of PCR fragments with short homology extensions.…”