The process of general recombination in Escherichia coli K-12

Summary A review of the data on the genetic determination of general recombination in Escherichia coli introduces three alternative pathways of recombination, RecBC, RecF, and RecBCF. One recBC-dependent pathway is functional in recF ^– cells. An initiating endonuclease is involved, acting on the chi-sites of DNA. The second is recF-dependent, acting in the double mutant recBC sbcB. The corresponding endonuclease uses the fre-sites as a substrate. A third pathway acting in wild-type cells is mixed. Both enzymatic systems participate in the overall process. We shall call it RecBCF.Using the thermosensitive recA44 mutant it became possible to study the kinetics of integration of donor DNA into the recipient chromosome via the RecF and RecBCF pathways of recombination. The RecF pathway is characterized by delayed recombination; not less than 14 h being needed to complete the process at 35° C. By the RecBCF pathway (wild-type recipient) the reaction is fast, as described by Lloyd and Johnson (1979). The two stage nature of the RecF pathway is important. First an intermediate product is formed during a short time interval. This product is resistant to the degrading exonuclease V. Afterward the intermediate product is slowly integrated into the recipient chromosome. Autoradiography of this intermediate product, extracted from exconjugants, shows that it consists of closed DNA circles. Their length is within the limits 2–15 min on the E. coli map. Their average value is in fair agreement with genetic estimations of the integrated DNA fragments.Taking into consideration the similarity between genetic determinations of the fre-effects and the heterogeneity of the progeny, we conclude that the intermediate structures formed contribute to this heterogeneity.     

Unequal genetic exchange in <Emphasis Type="Italic">Escherichia coli</Emphasis> tandem duplications may represent a special pathway of homologous recombination

Heterozygous tandem duplications that appear in Escherichia coli conjugation matings segregate different types of haploid and diploid recombinants formed by unequal crossing over between sister chromosomes. As shown previously, the frequency of segregants in the extended duplication D104 (150 kb or more than 3 min of the genetic map) heterozygous for E. coli deo-operon genes (deoA deoB::Tn5/deoC deoD) is not decreased in strains with defective RecBCD and RecF recombination pathways. Analysis of a shorter duplication of this type (46 kb) showed that the frequency of segregants in the strain recBC sbcBC recF was similar to that in a strain with undamaged system of recombination. Thus, genetic exchange between direct DNA repeats in tandem duplications may follow a special pathway of homologous recombination, which is independent of the recBC and recF genes.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 307–311.Original Russian Text Copyright © 2005 by Sukhodolets, Prokopev.     

Genetic determination of the donor properties in Escherichia coli K-12. Phenomena of chromosome mobilization and integrative suppression.

Summary The chromosome mobilization is the ability of F⁺ donors to introduce part of the chromosome besides F-plasmids into the recipient cells during conjugation. We studied the genetic determination of this phenomenon. Most efficient almost like true Hfr's are F⁺-cells of the genotype recBC ^- sbcB ^- belonging to the RecF-recombination type. Their ability to chromosome mobilization is 50 fold higher comparing with wild type F⁺ (of the RecBC-recombination type). This property is fully dependent on the recF gene but does not depend on recL. The donors recBC ^-F⁺ but without the mutation sbcB ^- act in mobilization about 4 times weaker than wild type. Hence we see two main levels of mobilization, quantitatively very different: a recF-dependent and recBC-dependent. Both reveal an absolute requirment of the product of recA gene.The efficiency of mobilization of different markers along the chromosome was studied and mapped. The maps were identical, in spite of great difference in absolute frequencies for the RecF- and Rec BC-pathways. They are not at all random. The sites of mobilization are coinsident with the points of interaction of the F-factor leading to stable Hfr's. Therefore it is suggested that these sites of predominant mobilization are IS-sequences and that during chromosome mobilization single-strand integration of the F-factor via a semichiasmus is effected. It gives a pulse to initiate DNA transfer into the recipient but is unstable and transient and does not yield true Hfr's.The suppression of the Dna^ts phenotype in F⁺ cells due to the integration of an F-plasmid into the chromosome (integrative suppression) is increased manyfold on the RecF-pathway of recombination. Probably it is a manifestation of mentioned hot spots of recombination.The regions fre described earlier (Bresler et al., 1978) and confirmed in this paper are regarded as substrates of some recF-dependent endonuclease of recombination. Probably they coinside with clusters of IS-sequences.     

Recombinational instability of F'' plasmids in Escherichia coli K-12: localization of fre-sites

Summary The F plasmids ORF-1 (purE ⁺ tsx ^s proC ⁺ lac ⁺) and F14 (agrE ⁺ metB ⁺ ilv ⁺) contain active regions of recombination, fre I and fre II correspondingly. The plasmid ORF-1 is stable in recF ^– cells. (i.e., with the RecBC pathway of recombination) and decays in rec ⁺ cells (RecBCF pathway) giving two types of product: F⁺ and plasmid pCK-1 (tsx ^s proC ⁺ lac ⁺) containing part of the initial DNA. They are extremely instable in the presence of the RecF pathway, (recBC ^– sbcB ^–), yielding F⁺ and plasmid pCK-2 (proC ⁺ lac ⁺). The instability of plasmids depends on a region of homology between the chromosome and the episome. The instability of ORF-1 shows the participation of IS3 elements (_{1 3} and _{3 1}) in the recA, recF-dependent recombinational decay and allows localization of two active sites on the chromosome: fre I1 between purE and tsx markers and fre I2 between tsx and proC.The plasmid F14, in accordance with published data, is able to yield F⁺ cells by recA-independent recombination. But eventually this plasmid may undergo a recA, recF-dependent decay. Genetic analysis of these events allows localization of an active point of recombination, fre II1, between argE and metB. Another active point is localized inside the F factor. The recA-dependent decay of plasmid F-14 is also excluded on the RecBC pathway (recF ^– strains).     

Tetracycline-resistance inEscherichia coli; a genetic contribution

A non-transmissible tetracycline-resistance plasmid inE. coli was found to be transmissible by transduction and by conjugation with the aid of theE. coli K12 sex-factor. Transfer of the tetracycline-resistance plasmid (R-tet) by transduction or conjugation to anE. coli K12 Hfr strain revealed that the plasmid was incompatible with the integrated F-factor. Selection for tetracycline-resistance after conjugation or transduction yielded Hfr colonies which carried the tetracycline-resistance determinant as a chromosomal marker. The tetracycline-resistance determinant was integrated at the 1 min region of theE. coli chromosome map (Taylor and Trotter, 1967) between the markersara andleu. Apart from Hfr colonies with a chromosomal tetracycline-resistance determinant, F-gal⁺-mediated transfer of R-tet to strain Hfr R4 gave some colonies in which the tetracycline-resistance determinant was carried on a fused plasmid that, besides the resistance determinant, contained thegal ⁺ marker of the original F-gal ⁺. This fused plasmid is transmissible and confers to an F⁻ cell male-specific phage-sensitivity, like an F-factor does. It is suggested that this fused plasmid, which is compatible with the integrated F-factor in the Hfr R4 cells, arose by recombination between F-gal ⁺ and R-tet.     

Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants

We have determined the nucleotide sequence of a 3.5 kb segment in the recF region of the Staphylococcus aureus chromosome. The gene order at this locus, dnaA-dnaN-recF-gyrB is similar to that found in the replication origin region of many other bacteria. S. aureus RecF protein (predicted molecular mass 42.3 kDa), has 57% amino acid sequence identity with the Bacillus subtilis RecF protein (42.2 kDa), but only 26% with the Escherichia coli RecF protein (40.5 kDa). We have shown that the S. aureus recF gene partially complements the defect of a B. subtilis recF mutant, but does not complement an E. coli recF strain. The amino acid sequence alignment of seven available RecF proteins (five of them from bacteria of gram-negative origin) allowed us to identify eight highly conserved regions ( to ) and to predict five new conserved regions within the gram-positive group (a to f). We suggest that the basic mechanism of homologous recombination is conserved among free-living bacteria.     

Role of the recF gene of Escherichia coli K-12 in lambda recombination

Summary When Escherichia coli K12() lysogens are infected with heteroimmune phage, which are unable to replicate, general recombination between phage and prophage depends on the bacterial recF gene. It has been shown that in E. coli K12 postconjugational recombination, the RecF pathway only works with full efficiency if exonuclease I is absent (Clark 1973). However, results presented in this paper indicate that under conditions in which replication is blocked, the recombination pathway dependant on the recF gene is fully active in producing viral recombinants even, if the phage is Red⁺, in the presence of exonuclease I. In contrast, removal of exonuclease and protein requires elimination of exonuclease I for an efficient RecF pathway. It is concluded that the Red system cooperates with the RecF pathway and that this cooperation involves overcoming the inhibitory effects of exonuclease I. In the absence of exonuclease, protein stimulates recF-dependent recombination but does not suffice to prevent the negative effect of exonuclease I. In the presence of protein, full efficiency of the RecF pathway can be obtained either via cooperation with exonuclease I or, if the viral exonuclease is defective, via inactivation of exonuclease I. Since activity of exonuclease appears necessary to overcome the inhibitory effects of exonuclease I, it is proposed here that exonuclease diverts material from the RecF pathway in a shunt reaction which allows completion of recF-initiated recombinational intermediates via a mechanism insensitive to exonuclease I.When replication is allowed, the Rec system produces viral recombinants mainly via a recF-independent mechanism. However, a major contribution of the RecF pathway to recombination is observed after removal of the Red system and exonuclease I.Obra social de la Caja de Ahorros de Valencia (Director: S. Grisolía)     

Effect of Mutations for the ruvABC Genes on Recombination between Direct DNA Repeats in Escherichia coli Strains Carrying Extended Tandem Duplications

The formation of haploid and diploid segregants was studied in Escherichia coli strains carrying heterozygous tandem duplications deoA deoB::Tn5/deoC deoD in the deoCABD operon region, in the genome of mutants forruvABCgenes. Homologous recombination in duplications of rec ⁺ strains and in recBC sbcB, recQand recF mutants, including those with blocks of both the RecBCD and RecF pathway, was shown in our previous work to be similar to adaptive mutagenesis: in this case, practically each cell forms a recombinant on a selective medium. In this work, mutants for ruv genes were found to differ in this respect, forming segregants at a frequency that was decreased by several orders of magnitude. These data confirm the conclusion that the genetic exchange in duplications proceeds through a special pathway of adaptive (or replicative) recombination connected with DNA replication. Upon selection of recombinants under conditions of thymine starvation, recombination cannot also be induced in ruv mutants. The recombinogenic effect of thymine starvation seems to occur at late stages of recombination, which are controlled by ruvABC genes.     

Genetic and physical mapping of recF in Escherichia coli K-12

Summary Two factor transductional crosses place recF at approximately 82 min on the E. coli chromosome; recF is highly cotransducible with dnaA and gyrB (cou). Transductional analysis with a series of tna specialized transducing phages carrying chromosomal DNA from the tnaA region place recF between dnaA and gyrB. This analysis also indicates that a gene lying in the same region and producing an easily detectable protein (estimated MW of 45 kD) is dnaN and not recF.     

Involvement of recF in 254 nm Ultraviolet Radiation Resistance in Deinococcus radiodurans and Escherichia coli

recF is a critical gene involved in the RecFOR pathway of DNA repair in Deinococcus radiodurans (D. radiodurans). To investigate the role of recF in UV-C radiation resistance, we generated the recF-deficient D. radiodurans strain R1, expressed recF in Escherichia coli (E. coli) BL21 cells, and compared the ability of each to resist UV-C radiation by F ₁₀, inactivation constant (IC), and extrapolation number (N). The mutation of recF in D. radiodurans R1 resulted in characteristic slow growth and dramatic sensitivity to UV-C irradiation. Transformation of recF into E. coli BL21 cells resulted in increased UV-C resistance compared to untransformed E. coli BL21 cells. These results suggested that recF is needed for the replication of D. radiodurans. Furthermore, as a part of the RecFOR pathway in D. radiodurans, disruption of recF could dramatically decrease the UV-C resistance of D. radiodurans and recF could increase the UV-C resistance of E. coli BL21 cells.     

Genetic Recombination in Klebsiella pneumoniae An Approach to Genetic Linkage Mapping

Genetic recombination was observed between two different strains of Klebsiella pneumoniae, which is a non-motile and encapsulated bacterium belonging to the family Enterobacteriaceae and has about 55% of its DNA content as GC. The mode of recombination seemed to be similar to that of the F-factor mediated conjugation in Escherichia coli. One strain acted as the donor and the other as the recipient, and a relatively large fragment of the donor's chromosome was transferred unilaterally and unidirectionally by cell to cell contact. No genetic factor which is associated with the recombination has been identified. The genetic linkage map of K. pneumoniae was analyzed various mutants derived from the two strains. It was found that the 28 markers so far investigated were arranged linearly in a single linkage group, and that the genetic linkage map of K. pneumoniae, like that of E. coli, could be considered circular. The proposed genetic linkage map of K. pneumoniae was quite similar to that of E. coli or Salmonella typhimurium. The close similarities in this map among the three species suggest a possibility that K. pneumoniae may have differentiated from an ancestor common all three species.     

Genetic Mapping of Cold Resistance Gene of Escherichia coli

Using cold resistant mutants, MET1 and MET2, obtained from Escherichia coli K-12, genetic mapping of the cold resistance gene(s) of E. coli was performed by the conjugation and transduction techniques. The gene(s) was confirmed to be located close to trpB at 28 min (revised chromosome linkage map, 1983) on the E. coli chromosome.     

Stimulation of recombination between homologous sequences on carcinogen-treated plasmid DNA and chromosomal DNA by induction of the SOS response in Escherichia coli K12

Summary Previous studies have shown that transformation of Escherichia coli by plasmid DNA modified in vitro by carcinogens leads to RecA-dependant recombination between homologous plasmid and chromosomal DNA sequences. The mechanism of this recombination has now been studied using recombination-deficient mutants, and the influence of induction of the SOS response on the level of recombination investigated. Plasmid pNO1523, containing the str ⁺ operon (Sm^s), has been modified in vitro by either irradiation with UV light, or by reaction with (±) trans-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) and used to transform streptomycin-resistant hosts. The formation of Amp^r transformants which also carry streptomycin resistance was used as a measure of the level of recombination between plasmid and chromosomal DNA. Transformation of recB and recC mutants produced no change in the level of recombination while in the recF mutant a significant decrease was observed compared to the wild type host. Thermal induction of the SOS response in tif-1 and tif-1 umuC mutants followed by transformation led to a four-fold increase in recombination in both cases. The results suggest that the streptomycin-resistant transformants arise exclusively via a recombinational pathway which is largely dependant on the recF gene product, and that this pathway is influenced by induction of the SOS response. These results are discussed in terms of the mechanism of this recombination.     

A mutation of Streptomyces lividans which prevents intraplasmid recombination has no effect on chromosomal recombination

Summary A mutation (rec-46) of Streptomyces lividans, previously shown to prevent (or greatly diminish) homologous and illegitimate intraplasmid recombination, was shown to have no effect on generalised chromosomal recombination occurring in matings or in protoplast fusions, nor to affect homologous recombination between a recombinant plasmid and the host chromosome. By comparison with Escherichia coli mutants defective in various aspects of recombination, the rec-46 mutation is similar to those in recF, recJ, recO and topA.     

Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants

We have determined the nucleotide sequence of a 3.5 kb segment in the recF region of the Staphylococcus aureus chromosome. The gene order at this locus, dnaA-dnaN-recF-gyrB is similar to that found in the replication origin region of many other bacteria. S. aureus RecF protein (predicted molecular mass 42.3 kDa), has 57% amino acid sequence identity with the Bacillus subtilis RecF protein (42.2 kDa), but only 26% with the Escherichia coli RecF protein (40.5 kDa). We have shown that the S. aureus recF gene partially complements the defect of a B. subtilis recF mutant, but does not complement an E. coli recF strain. The amino acid sequence alignment of seven available RecF proteins (five of them from bacteria of gram-negative origin) allowed us to identify eight highly conserved regions (α to θ) and to predict five new conserved regions within the gram-positive group (a to f). We suggest that the basic mechanism of homologous recombination is conserved among free-living bacteria.     

A genetic assay for transversion mutations in bacteriophage T4

Summary Seventy-two mutants deficient in formate-nitrate reductase activity were selected in Escherichia coli strain PK 27, by two different procedures. Forty-five strains were selected on the basis of chlorate resistance and 27 strains were selected by their inability to reduce nitrate with formate as an electron donor. Genetic analysis of these strains showed that the two techniques yield distinctly different distributions of mutants among the various controlling genetic loci. Chlorate resistance appears to select for severe alterations in the nitrate reductase system; 98% of these mutants fell into the pleiotropic chl A, B, D and E classes and are deficient in all the activities of the formate-hydrogenlyase pathway as well as formate-nitrate reductase pathway. In contrast, 48% of the mutants selected for their inability to reduce nitrate with formate as the electron donor were of the chl C class and two new classes were identified among mutants selected by this procedure. Chl F mutants are linked to tryptophan and the chl C locus. Chl G mutants map at zero minutes on the E. coli genetic map.     

Isolation and genetic mapping of Escherichia coli aminopeptidase mutants

Summary Many mutant strains devoid of aminopeptidase activity have been isolated in Escherichia coli. All of them produce cross-reacting material when tested against specific antiaminopeptidase antibody. The map position of the locus specifying this enzyme has been determined by three conjugations and two P₁ mediated transduction experiments. By analogy with Salmonella typhimurium this locus has been called pepN (Miller, 1975). Mutations in pepN are jointly transduced with fabA and pyrD at high frequency. These data and conjugation results suggest a location between 20.5 and 22.5 minutes on E. coli genetic map.     

P1 transduction map spanning the replication terminus of Escherichia coli K12

Summary The region of the E. coli chromosome that contains the replication terminus has not previously been spanned by P1 cotransduction. We have used Tn5, Tn9 and Tn10 transposons inserted in this region as genetic markers, and have constructed a genetic map that extends from fnr (min 29.3) to manA (min 35.7). The relevant transposons that have been mapped in this region and which are described in this report are trgl::Tn5 (min 31.1), zdc-235::Tn10 (min 32.3), zdd-230::Tn9 (min 33.3), and zde-234::Tn10 (min 34.2). The size of this region as determined by P1 cotransduction is very similar to previous estimates obtained by bacterial conjugation.     

Interplasmidic and intraplasmidic recombination in Escherichia coli K-12

Summary The construction of plasmids which facilitate the study of interplasmidic and intraplasmidic recombination is described. In this system, a single recombination event between two mutated Te^r genes on separate plasmids or on one plasmid leads to a change in the host phenotype from sensitivity to resistance to tetracycline.Recombination proficiencies have been determined for different E. coli K-12 strains: both interplasmidic and intraplasmidic recombination are independent of the recBC gene product. RecA mutations decrease the proficiency of plasmidic recombination 40–100 fold. Intraplasmidic and interplasmidic recombination via the recE pathway are more efficient than via the recBC pathway. Intraplasmidic recombination, but not interplasmidic recombination via the recE pathway is independent of a functional recA product.     

Genetic analysis of the recF pathway to genetic recombination in Escherichia coli k12: Isolation and characterization of mutants

A recombination proficient strain ofEscherichia coli which is recB^? recC^? sbcB^? has been subjected to mutagenesis by nitrosoguanidine. Among the recombination deficient mutants isolated one was sbcB⁺, three were recA^～ and 11 were mutants in at least four newrec genes: recF, recJ, recK and recL. recF143 and recL152 are cotransducible with ilv but they lie on opposite sides of the ilv operons as determined by F$\text{\$}\text{?}$studies. recF, recL and recK are not involved in the RecBC pathway of recombination since a recB⁺recC⁺sbcB^? strain carrying a mutation in one of these genes is recombination proficient. Hence the hypothesis that a RecF pathway of recombination can operate as a partially independent substitute for the RecBC pathway of recombination is supported. recF^?recB⁺ and recF⁺recB^? single mutants are sensitive to u.v. irradiation while the recF^?recB^? double mutant is more sensitive than either single mutant. The sensitivity of the recB^?recC^?sbcB^?recF^? strain approaches the sensitivity of a recA^? single mutant. This is interpreted to mean that there are partially independent RecF and RecBC pathways for the repair of u.v. damage. recJ and mutations were not mapped precisely; hence the mutant properties they confer can not be stated conclusively.