Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding.

Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype. In wild-type cells, 49% of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble. Remarkably, in recD cells about 25% of the DNA was rendered acid soluble. The DNA degradation in recD cells depended on intact recB and recC genes. The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) or xonA (which abolishes the 3' single-strand-specific exonuclease I). In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant. Results similar to those with the set of recD strains were also obtained with a recC++ mutant (in which the RecD protein is intact but does not function) and its recJ, xonA, and recJ xonA derivatives. The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation. It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants. The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway.     

Gene Replacement with Linear DNA Fragments in Wild-Type Escherichia Coli: Enhancement by Chi Sites

During conjugation and transduction of Escherichia coli even numbers of recombinational exchanges are required for replacement of a gene on the circular chromosome. We studied gene replacement using a related method of gene transfer (transformation with 6.5-kb linear DNA fragments) as an experimental model for conjugation and transduction. Two properly situated Chi sites, 5' GCTGGTGG 3', stimulated gene replacement ~50-fold, more than the sum of the stimulation by the individual Chi sites. Gene replacement was dependent on RecA and RecB functions. Similar results were obtained with an alternative experimental model in which linear DNA fragments were generated from phage λ by intracellular EcoRI restriction following infection. Dual Chi site-stimulation of these RecA-, RecB-dependent recombination events thus did not depend upon the mode of delivery of the linear DNA into the cells. A single DNA fragment with two Chi sites was sufficient for gene replacement. These results support a one Chi-one exchange hypothesis (``long chunk' gene replacement), stemming from studies with purified RecBCD enzyme, and argue against models in which Chi converts RecBCD enzyme to a state capable of promoting multiple exchanges on one DNA molecule. These results also provide a method for gene targeting in wild-type E. coli and suggest a method for gene targeting in other organisms.     

Exchange of chromosomal and plasmid alleles in Escherichia coli by selection for loss of a dominant antibiotic sensitivity marker.

Transfer of an allele from a donor DNA to a recipient DNA molecule was selected by the loss of a dominant conditional lethal mutation previously incorporated ito the gene of interest in the recipient DNA. Both the Escherichia coli chromosome and plasmids carrying E. coli genes were used successfully as donor molecules. Recipient molecules for these exchanges were constructed in vitro by using the rpsL gene, which confers sensitivity to streptomycin, to replace segments of specific E. coli genes located either on multicopy plasmids or in the E. coli chromosome. Plasmids carrying such replacements were capable of acquiring chromosomal alleles of the gene(s) of interest, and strains carrying rpsL replacements in the chromosome were capable of acquiring plasmid-encoded alleles at the sight of the rpsL replacement. In both situations, these allele transfers resulted in loss of the rpsL gene from the recipient DNA molecule. The desired transfer events constituted a large percentage of these events, which gave rise to viable colonies when appropriate donor-recipient pairs were subjected to streptomycin selection. Thus, this is a useful approach for transferring alleles of interest from plasmids to the E. coli chromosome and vice versa.     

Ectopic Integration of Transforming DNA Is Rare among Neurospora Transformants Selected for Gene Replacement

In a variety of organisms, DNA-mediated transformation experiments commonly produce transformants with multiple copies of the transforming DNA, including both selected and unselected molecules. Such ``cotransformants' are much more common than expected from the individual transformation frequencies, suggesting that subpopulations of cells, or nuclei, are particularly competent for transformation. We found that Neurospora crassa transformants selected for gene replacement at the am gene had not efficiently incorporated additional DNA, suggesting that nuclei that undergo transformation by homologous recombination are not highly competent at integration of DNA by illegitimate recombination. Spheroplasts were treated with DNA fragments homologous to am and with an Escherichia coli hph plasmid. Transformants were initially selected for hph (hygromycin(R)), allowed to conidiate to generate homokaryons and then selected for either Am(-) (gene replacements) or hph. Surprisingly, most am replacement strains were hygromycin(S) (124/140) and carried no extraneous DNA (116/140). Most transformants selected for hph also had ectopic copies of am DNA and/or multiple copies of hph sequences (32/35), generally at multiple sites, confirming that efficient cotransformation could occur. To test the implication that cotransformation involving gene replacement and ectopic integration is rare, we compared the yields of am replacement strains with or without prior selection for hph. The initial selection did not appreciably help (or hinder) recovery of strains with replacements.     

Gene 1.2 protein of bacteriophage T7. Effect on deoxyribonucleotide pools

The gene 1.2 protein of bacteriophage T7, a protein required for phage T7 growth on Escherichia coli optA1 strains, has been purified to apparent homogeneity and shown to restore DNA packaging activity of extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants (Myers, J. A., Beauchamp, B. B., White, J. H., and Richardson, C. C. (1987) J. Biol. Chem. 262, 5280-5287). After infection of E. coli optA1 by T7 gene 1.2 mutant phage, under conditions where phage DNA synthesis is blocked, the intracellular pools of dATP, dTTP, and dCTP increase 10-40-fold, similar to the increase observed in an infection with wild-type T7. However, the pool of dGTP remains unchanged in the mutant-infected cells as opposed to a 200-fold increase in the wild-type phage-infected cells. Uninfected E. coli optA+ strains contain severalfold higher levels of dGTP compared to E. coli optA1 cells. In agreement with this observation, dGTP can fully substitute for purified gene 1.2 protein in restoring DNA packaging activity to extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants. dGMP or polymers containing deoxyguanosine can also restore packaging activity while dGDP is considerably less effective. dATP, dTTP, dCTP, and ribonucleotides have no significant effect. The addition of dGTP or dGMP to packaging extracts restores DNA synthesis. Gene 1.2 protein elevates the level of dGTP in these packaging extracts and restores DNA synthesis, thus suggesting that depletion of a guanine deoxynucleotide pool in E. coli optA1 cells infected with T7 gene 1.2 mutants may account for the observed defects.     

Constitutive inhibition of DNA degradation due to the enzyme RecBCD in the radiation-resistant Escherichia coli K-12 mutant Gam(r)444]

Exonucleolytic degradation of [3]H-labeled DNA was examined in partially purified fractions of lysates obtained from nonirradiated RecBCD enzyme-containing cells of Escherichia coli and in the radiation-resistant mutant Gamr444. The degradative activity was shown to be lowered in these cells to the same extent as in the recBC mutant. The efficiency of plating of the mutant phage T4 2-, DNA of which can be degraded by exonuclease V, was 400-fold higher on the strain Gamr444 than on the wild-type strain AB1157. This value was shown to be only twice as low as that on the recB mutant or on the strain AB1157 carrying plasmid pGam26 with a radiation-resistance allele gam26 cloned from mutant Gamr444. The data obtained confirmed the hypothesis that the Gamr444 mutant contains a constitutive inhibitor of exonucleolytic activity of the RecBCD enzyme in nonirradiated cells. This inhibitor was shown to be encoded by the gam26 allele that had previously been mapped at 56.8 min of the E. coli chromosome. A possible mechanism of the involvement of this inhibitor in enhanced radiation resistance of the mutant Gamr444 is considered.     

Mechanism of intramolecular recyclization and deletion formation following transformation of Escherichia coli with linearized plasmid DNA.

The deletion end-points of a number of type I (less than monomeric) plasmid deletants obtained by transforming recA+ or recA- E. coli with linear pBR322 DNA were determined by DNA sequencing. In both monodirectional and bidirectional deletions the recyclization point was normally characterized by recombination between directly repeated sequences of between 4 and 10 bp present on each arm of the linearized pBR322 molecule. Frequently, short tracts of uninterrupted homology involved in recombinational recircularization were embedded in regions of relative non-homology. A model predicting the probability of matching sequences in either end of a linear plasmid molecule is presented. It is proposed that exonucleolytic processing of the exposed termini of linear plasmid molecules generates substrates for subsequent recombinational recyclization and deletion. The activity of host recombination and repair functions in recircularizing linear DNA molecules explains the generation of many of the aberrant recombinant DNA constructs obtained during gene cloning procedures.     

Red recombinase assisted gene replacement in Klebsiella pneumoniae

The Red recombinase system, the most convenient genetic tool applied in Escherichia coli and other bacteria, was introduced for gene replacement in Klebsiella pneumoniae. The novel K. pneumoniae gene replacement system comprised the Red and FLP recombinases expression vector pDK6-red and pDK6-flp, and linear DNA fragments which encompassed a selective marker gene with target gene flanking extensions; the latter were PCR amplified using a plasmid DNA template obtained by in vivo recombination in E. coli. In this study, dhak1 gene, encoding a subunit of dihydroxyacetone kinase II, was deleted markerlessly at a transformation ratio of 260 CFU/μg DNA, i.e., 1,000-fold higher than that achieved in the native way. Our studies provide an efficient method with detailed protocol to perform gene replacement in K. pneumoniae and has great potential to be developed as a routine genetic approach for this important industrial microorganism.     

Synthesis by DNA polymerase I on bleomycin-treated deoxyribonucleic acid: a requirement for exonuclease III

phi X174 RFI DNA treated with bleomycin (BLM) under conditions permitting nicking does not serve as a template-primer for Escherichia coli DNA polymerase I. Purified exonuclease III from E. coli and extracts from wild-type E. coli strains are able to convert the BLM-treated DNA to suitable template-primer, but extracts from exonuclease III deficient strains are not. Brief digestion by exonuclease III is enough to create the template-primer, suggesting that the exonuclease III is converting the BLM-treated DNA by a modification of 3' termini. The exonucleolytic rather than the phosphatase activity of exonuclease III appears to be involved in the conversion. Comparative studies with micrococcal nuclease indicate that BLM-created nicks do not have a simple 3'-P structure. Bacterial alkaline phosphatase does not convert BLM-treated DNA to template-primer. The endonuclease VI activity associated with exonuclease III does not incise DNA treated with BLM under conditions not allowing nicking, in contrast to DNA with apurinic sites made by acid treatment, arguing that conversion does not require the endonuclease VI action on uncleaved sites.     

Different nuclease activities in competent and noncompetent Bacillus subtilis.

Competent and noncompetent cells of Bacillus subtilis were separated on the basis of their different buoyant densities. The two types of cells were compared with respect to their interactions with exogenous deoxyribonucleic acid(DNA). After exposure of DNA to the cells, the unadsorbed fraction of DNA molecules was examined. Both types of cells decreased the biological activity of this DNA, the inactiviation exerted by noncompetent cells being more severe than that exerted by competent cells. Sedimentation analysis of the inactivated DNA revealed that fragments of DNA are produced, owing mainly to the introduction of double-strand scissions. In addition to this fragmentation, the competent bacteria extensively digested the DNA exonucleolytically. This type of breakdown was specifically related to the competent state rather than to the state of low density. The exonucleolytic activity is, in all probability, associated with the cell envelope, because most of the activity is released into the medium when the cells are converted to protoplasts. At 37 C the competence-specific exonucleolytic breakdown started 2 to 3 min after the binding of DNA to the cells. In unfractionated cultures, breakdown may proceed until 70% of the total amount of DNA added has been made acid soluble. Nontransforming Escherichia coli DNA was also subject to exonucleolytic degradation; it seems unlikely,therefore, that this type of breakdown occurs as a consequence of recombination. Since ethylenediaminetetraacetate blocked both transformation by native DNA and the exonucleolytic breakdown of bound DNA, we suggest that the breakdown of DNA by competent cells fulfills an essential function in genetic transformation of B. subtilis.     

A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation

A rapid microcentrifuge-based method is described for preparation of Pseudomonas aeruginosa electrocompetent cells with up to 10,000-fold increased transformation efficiencies over existing procedures. This increased efficiency now enables the use of transformation for all applications requiring DNA transfer. These include transfer of chromosomal mutations marked with antibiotic resistance genes between P. aeruginosa strains, which solves the riddle of not having an efficient and reliable transduction procedure for this bacterium. Not surprisingly, the method also allows for very efficient transformation with replicative plasmids, with transformation efficiencies ranging from 10(7) to >10(11) transformants per microgram of DNA. Lastly, with efficiencies of up to >10(3) transformants per microgram of DNA the method replaces in most instances conjugation for the transfer of non-replicative plasmids used in gene replacement, site-specific gene integration and transposon mutagenesis experiments.     

Homologous pairing in genetic recombination. Purification and characterization of Escherichia coli recA protein

RecA protein, which is essential for genetic recombination in Escherichia coli, was extensively purified from a strain of E. coli which contained the recA gene cloned in a plasmid (Sancar, A., and Rupp, W. D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3144-3148). Using the DNA-dependent ATPase activity of recA protein as an assay, we obtained about 60 mg of purified recA protein from 100 g of cells. Ten micrograms or 1 microgram of the purified protein exhibited only one detectable band with Mr approximately = 40,000 upon sodium dodecyl sulfate-acrylamide gel electrophoresis. More than 99% of the ATPase activity of purified recA protein was dependent on single-stranded DNA. Purified recA protein had no detectable DNase, topoisomerase, or ligase activities. The enzyme was stable for a least a year when stored at 0-4 degrees C. The half-life of the ATPase activity of 25 microM recA protein was 37 min at 51 degrees C. Purified recA protein binds to single-stranded and double-stranded DNA, unwinds duplex DNA by a mechanism that is stimulated by single-stranded DNA or oligonucleotides, and pairs homologous single strands with duplex DNA.     

Role of Gene 52 in Bacteriophage T4 DNA Synthesis

In an attempt to elucidate the mechanism of delayed DNA synthesis in phage T4, Escherichia coli B cells were infected with H17 (an amber mutant defective in gene 52 possessing a "DNA-delay" phenotype). The fate of (14)C-labeled H17 parental DNA after infection was followed: we could show that this DNA sediments more slowly in neutral sucrose than wild-type DNA 3 min postinfection. In pulse-chase experiments progeny DNA was found to undergo detachment from the membrane at 12 min postinfection. Reattachment to the membrane was found to be related to an increase in rate of DNA synthesis. A nucleolytic activity that is absent from cells infected by wild-type phage and from uninfected cells could be detected in extracts prepared from mutant-infected cells. In contrast, degradation of host DNA was found to be less extensive in am H17 compared with wild-type infected cells. Addition of chloramphenicol to mutant-infected cells 10 min postinfection inhibited the appearance of a nuclease activity on one hand and suppressed the "DNA-delay" phenotype on the other hand. We conclude that the gene 52 product controls the activity of a nuclease in infected cells whose main function may be specific strand nicking in association with DNA replication. This gene product might directly attack both E. coli and phage T4 DNA, or indirectly determine their sensitivity to degradation by another nuclease.     

Mutational inactivation of the Saccharomyces cerevisiae RAD4 gene in Escherichia coli.

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. When plasmids containing the wild-type gene were transformed into various Escherichia coli strains, transformation frequencies were drastically reduced. Most plasmids recovered from transformants showed deletions or rearrangements. A minority of plasmids recovered from E. coli HB101 showed no evidence of deletion or rearrangement, but when they were transformed into S. cerevisiae on centromeric vectors, little or no complementation of the UV sensitivity of rad4 mutants was observed. Deliberate insertional mutagenesis of the wild-type RAD4 allele before transformation of E. coli restored transformation to normal levels. Plasmids recovered from these transformants contained an inactive rad4 allele; however, removal of the inserted DNA fragment restored normal RAD4 function. These experiments suggest that expression of the RAD4 gene is lethal to E. coli and show that lethality can be prevented by inactivation of the gene before transformation. Stationary-phase cultures of some strains of E. coli transformed with plasmids containing an inactivated RAD4 gene showed a pronounced delay in the resumption of exponential growth, suggesting that the mutant (and, by inference, possibly wild-type) Rad4 protein interferes with normal growth control in E. coli. The rad4-2, rad4-3, and rad4-4 chromosomal alleles were leaky relative to a rad4 disruption mutant. In addition, overexpression of plasmid-borne mutant rad4 alleles resulted in partial complementation of rad4 strains. These observations suggest that the Rad4 protein is relatively insensitive to mutational inactivation.     

A DNA sequence-specific electrochemical biosensor based on alginic acid-coated cobalt magnetic beads for the detection of E. coli

A new type of DNA sequence-specific electrochemical biosensor based on magnetic beads for the detection of Escherichia coli is reported in the present work. Alginic acid-coated cobalt magnetic beads, capped with 5'-(NH(2)) oligonucleotide and employed not only for magnetic separation but also as the solid adsorbent, were used as DNA probes to hybridize with the target E. coli DNA sequence. This assay was specific for E. coli detection depending on the uid A gene, which encodes for the enzyme β-d-glucuronidase produced by E. coli strains. When daunomycin (DNR) was used as DNA hybridization indicator, the target sequences of E. coli hybridized with the probes resulted in the decrease of DNR reduction peak current, which was proportional to the E. coli concentration. The optimization of the hybridization detection was carried out and the specificity of the probes was also demonstrated. This DNA biosensor can be employed to detect a complementary target sequence for 3.0×10(-10) mol/L and denatured PCR products for 0.5 ng/μL. The linear range of the developed biosensor for the detection of E. coli cells was from 1.0×10(2) to 2.0×10(3) cells/mL with a detection limit of 50 cells/mL. After a brief enrichment process, a concentration of 10 cells/mL E. coli in real water samples was detected by the electrochemical biosensor.     

Natural plasmid transformation in<Emphasis Type="Italic">Escherichia coli</Emphasis>

Although Escherichia coli does not have a natural transformation process, strains of E. coli can incorporate extracellular plasmids into cytoplasm 'naturally' at low frequencies. A standard method was developed in which stationary phase cells were concentrated, mixed with plasmids, and then plated on agar plates with nutrients which allowed cells to grow. Transformed cells could then be selected by harvesting cells and plating again on selective agar plates. Competence developed in the lag phase, but disappeared during exponential growth. As more plasmids were added to the cell suspension, the number of transformants increased, eventually reaching a plateau. Supercoiled monomeric or linear concatemeric DNA could transform cells, while linear monomeric DNA could not. Plasmid transformation was not related to conjugation and was recA-independent. Most of the E. coli strains surveyed had this process. All tested plasmids, except pACYC184, could transform E. coli. Insertion of a DNA fragment containing the ampicillin resistance gene into pACYC184 made the plasmid transformable. By inserting random 20-base-pair oligonucleotides into pACYC184 and selecting for transformable plasmids, a most frequent sequence was identified. This sequence resembled the bacterial interspersed medium repetitive sequence of E. coli, suggesting the existence of a recognition sequence. We conclude that plasmid natural transformation exists in E. coli.     

Genetic transformation of various species of Enterococcus by electroporation

A transformation system for Enterococcus faecalis was developed which uses untreated (i.e., non-protoplasted) cells and the electroporation technique. The optimized protocol resulted in transformation efficiencies of up to 4 x 10(6) transformants per microgram of plasmid DNA. All strains of E. faecalis tested could be transformed by this method, albeit with differing transformation efficiencies. Using the protocol optimized for E. faecalis we successfully transformed Enterococcus faecium, E. hirae, E. malodoratus and E. mundtii.     

Involvement of recA and exr Genes in the In Vivo Inhibition of the recBC Nuclease

When Escherichia coli cells are gamma irradiated they degrade their deoxyribonucleic acid (DNA). The DNA of previously gamma-irradiated T4 phage is also degraded in infected cells. The amount of degradation is not only dependent on the dose but also on the genotype of the cell. The amount of degradation is less in cells carrying a recB or a recC mutation, suggesting that most of the DNA degradation is due to the recB(+) and recC(+) gene product (exonuclease V). In some strains a previous dose of ultraviolet (UV) light followed by incubation renders the cells resistant to DNA degradation after gamma irradiation. We have shown this inhibition to take place for infecting T4 phage also. By using six strains of E. coli selected for mutations in the genes recA, exr (or lex), and uvrB, we have been able to show that the preliminary UV treatment produces no change in recA and exr cells for both endogenous DNA degradation and the degradation of infecting irradiated T4 phage DNA, i.e., inhibition was not detected in these strains. On the other hand, wild-type cells and strains carrying mutations of uvrB show inhibition in both types of experiments. Because the recA gene product and the exr(+) (lex(+)) gene product are necessary for the induction of prophage, it is possible that the phenomenon of inducible inhibition requires recA(+) and exr(+) presence. One interpretation of these results is that an inducible inhibitor may be controlled by the exr gene.     

Endonuclease A degrades chromosomal and plasmid DNA of Escherichia coli present in most preparations of single stranded DNA from phagemids.

With E. coli, large and variable amounts of chromosomal and plasmid DNAs are observed in the supernatants of overnight cultures when the cells carry an endA mutation, but are not detected by gel electrophoresis when the cells carry the wild type allele of endA. Significant amounts of nuclease activity in DH11S endA+ supernatants were detected by two simple assays; the rapid degradation of added pBR322 plasmid DNA, as judged by agarose gel electrophoresis, and a decrease of more than 100000 fold in transformation efficiency of the added pBR322 plasmid DNA. By employing isogenic endA mutant and wild type strains of DH11S and DH10B/F' proAB+ laclq Z delta M15, it was shown that detectable levels of chromosomal and plasmid DNAs are observed only in the endA mutant strains. These results indicate that Endonuclease I activity is responsible for degradation of chromosomal and plasmid DNA usually present in preparations of ssDNA. Therefore, a wild type endA gene is useful for the rapid and simple production of highly purified ssDNA from cells containing phagemid vectors.