Suppression of recA deficiency in plasmid recombination by bacteriophage lambda beta protein in RecBCD- ExoI- Escherichia coli cells.

Plasmid recombination, like other homologous recombination in Escherichia coli, requires RecA protein in most conditions. We have found that the plasmid recombination defect in a recA mutant can be efficiently suppressed by the beta protein of bacteriophage lambda. beta protein is required for homologous recombination of lambda chromosomes during lytic phage growth in a recA host and is known to have a strand-annealing activity resembling that of RecA protein. The bioluminescence recombination assay was used for genetic analysis of beta-protein-mediated plasmid recombination. Efficient suppression of the recA mutation by beta protein required the absence of the E. coli nucleases exonuclease I and RecBCD nuclease. These nucleases inhibit a RecA-mediated plasmid recombination pathway that is more efficient than the pathway functioning in wild-type cells. Like RecA-mediated plasmid recombination in RecBCD- ExoI- cells, beta-protein-mediated plasmid recombination depended on concurrent DNA replication and on the activity of the recQ gene. However, unlike RecA-mediated plasmid recombination, beta-protein-mediated recombination in RecBCD- ExoI- cells was independent of recF and recJ activities. We propose that inactivation of exonuclease I and RecBCD nuclease stabilizes a recombination intermediate that is involved in RecA- and beta-protein-catalyzed homologous pairing reactions. We suggest that the intermediate may be linear plasmid DNA with a protruding 3' end, since these nucleases are known to interfere with the synthesis of such linear forms. The different recF and recJ requirements for beta-protein-dependent and RecA-dependent recombinations imply that the mechanisms of formation or processing of the putative intermediate differ in the two cases.     

Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant.

A recombinant plasmid carrying the recA gene of Aeromonas caviae was isolated from an A. caviae genomic library by complementation of an Escherichia coli recA mutant. The plasmid restored resistance to both UV irradiation and to the DNA-damaging agent methyl methanesulfonate in the E. coli recA mutant strain. The cloned gene also restored recombination proficiency as measured by the formation of lac+ recombinants from duplicated mutant lacZ genes and by the ability to propagate a strain of phage lambda (red gam) that requires host recombination functions for growth. The approximate location of the recA gene on the cloned DNA fragment was determined by constructing deletions and by the insertion of Tn5, both of which abolished the ability of the recombinant plasmid to complement the E. coli recA strains. A. caviae recA::Tn5 was introduced into A. caviae by P1 transduction. The resulting A. caviae recA mutant strain was considerably more sensitive to UV light than was its parent. Southern hybridization analysis indicated that the A. caviae recA gene has diverged from the recA genes from a variety of gram-negative bacteria, including A. hydrophila and A. sobria. Maxicell labeling experiments revealed that the RecA protein of A. caviae had an Mr of about 39,400.     

Enzymatic formation of biparental figure-eight molecules from plasmid DNA and their resolution in E. coli

A key intermediate in general genetic recombination is a structure in which two double-stranded DNA molecules are covalently linked by a single-strand crossover characteristic of a Holliday junction. When the DNA molecules are circular, the recombinant structures take the form of a figure eight. We have used purified E. coli enzymes to construct biparental figure-eight DNA molecules in vitro from the DNA of two partially homologous plasmids. When purified figure-eight structures are transfected into recA- E. coli cells, they are resolved to produce monomeric or dimeric plasmid progeny, apparently by the cutting and joining of the Holliday crossover. The maturation of figure-eight molecules in bacteria is characterized by the formation and recovery of both parental and recombinant types, cross-over at a frequency of up to 50% and the capability for mismatch repair at regions of hybrid DNA. In these three regards, the products of figure-eight maturation resemble recombinant chromosomes formed at meiosis. These observations show that biparental figure eights behave as recombination intermediates that can be resolved into mature recombinants without need for a functional recA+ gene product.     

Cloning and expression in Escherichia coli of a recA-like gene from the acidophilic autotroph Thiobacillus ferrooxidans

A recombinant plasmid, pRSR100, containing the functional analogue of the Escherichia coli recA gene was isolated from a genomic library of Thiobacillus ferrooxidans ATCC 33020. The plasmid complemented defects in DNA repair and homologous recombination in E. coli recA mutant strains. Antiserum raised against E. coli RecA protein reacted with the native but defective E. coli HB101 RecA protein; it did not react with protein extracts from the recA deletion mutant E. coli JK696, but it reacted with two protein bands in extracts of E. coli JK696(pRSR100). A single band with an apparent Mr equal to the higher-Mr band in E. coli JK696(pRSR100) was detected in T. ferrooxidans cell extracts with the E. coli RecA antiserum.     

Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis. II. Transfer of sequences propagated in Escherichia coli to B. subtilis

Recombinant plasmid DNA cloned in E. coli via the bifunctional vector pDH5060 suffered deletions when returned to B. subtilis. However, DNA preparations of identical chimeras containing homologous or heterologous sequences stably transformed B. subtilis at high efficiency when isolated from B. subtilis. The vector pDH5060, however, was not affected and could be stably shuttled between E. coli and B. subtilis at high frequency. These problems affected the transfer of clone pools and individual chimeras, irrespective of the restriction or recombination phenotype of B. subtilis recipients. Deleted chimeras lost at least one end of cloned inserts, and in most cases, flanking plasmid sequences. Single plasmid forms (intact or deleted) were isolated from several hundred individual Cmr-transformants this suggests that events leading to deletion of chimeric plasmid DNA occur during transformation by restriction of unmodified insert sequences propagated in the intermediate host, E. coli. This conclusion is discussed with regard to the mechanism of plasmid transformation in B. subtilis.     

Cloning and characterization of recA genes froM Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r

The recA genes of Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r have been isolated and introduced into Escherichia coli K-12. All the heterologous genes restore resistance to killing by UV irradiation and the mutagen 4-nitroquinoline-1-oxide in RecA- E. coli K-12 hosts. Recombination proficiency is also restored as measured by formation of Lac+ recombinants from duplicated mutant lacZ genes and the ability to propagate phage lambda derivatives requiring host recombination functions for growth (Fec-). The cloned heterologous genes increase the spontaneous induction of lambda prophage in lysogens of a recA strain. Addition of mitomycin C stimulates phage production in cells carrying the E. coli B/r and S. flexneri recA genes, but little or no stimulation is seen in cells carrying the E. carotovora and P. vulgaris recA genes. After treatment with nalidixic acid, the heterologous RecA proteins are synthesized at elevated levels, a result consistent with their regulation by the E. coli K-12 LexA repressor. Southern hybridization and preliminary restriction analysis indicate divergence among the coding sequences, but antibodies prepared against the E. coli K-12 RecA protein cross-react with the heterologous enzymes, indicating structural conservation among these proteins.     

Transgenic expression of RecA of the spirochetes Borrelia burgdorferi and Borrelia hermsii in Escherichia coli revealed differences in DNA repair and recombination phenotypes

After unsuccessful attempts to recover a viable RecA-deficient mutant of the Lyme borreliosis agent Borrelia burgdorferi, we characterized the functional activities of RecA of B. burgdorferi, as well as RecA of the relapsing fever spirochete Borrelia hermsii and the free-living spirochete Leptospira biflexa, in a recA mutant of Escherichia coli. As a control, E. coli RecA was expressed from the same plasmid vector. DNA damage repair activity was assessed after exposure of the transgenic cells to UV light or the radiomimetic chemicals methyl methanesulfonate and mitomycin C. Recombination activity in the cells was assessed by using an assay for homologous recombination between repeats in the chromosome and by measuring the ability of the cells to foster lytic growth by red gam mutant bacteriophage lambda. Overall, we found that transgenic cells with recA genes of B. burgdorferi, B. hermsii, and L. biflexa had approximately equivalent activities in promoting homologous recombination in the lacZ duplication assay, but cells with B. burgdorferi recA and, most notably, B. hermsii recA were significantly less capable than cells with L. biflexa recA or E. coli recA in responding to DNA damage or in facilitating plaque formation in the phage assay. The comparatively poor function of Borrelia recA in the latter set of assays may be the consequence of impaired coordination in the loading of the transgenic RecA by RecBCD and/or RecFOR in E. coli.     

Site-specific recombination between cloned attP and attB sites from the Haemophilus influenzae bacteriophage HP1 propagated in recombination-deficient Escherichia coli.

Plasmids were constructed which contain both attP and attB DNA segments derived from the insertion sites of the lysogenic bacteriophage HP1 and its host, Haemophilus influenzae. Similar plasmids containing the two junction segments (attL and attR regions) between the phage genome and the lysogenic host chromosome were also prepared. The formation of recombinant dimer plasmids was observed when attP-attB plasmids were propagated in Escherichia coli HB101 (recA), while plasmids containing the junction segments did not form recombinant dimers. Deletion of the phage DNA segment adjacent to the attP site from the attP-attB constructions eliminated detectable recombination, suggesting that this sequence contains the gene encoding the HP1 integrase. No plasmid recombination was observed in strains of E. coli defective in integration host factor. This suggests that integration host factor is important in the expression or activity of the system which produces the site-specific recombination of sequences derived from HP1 and H. influenzae. Further, it suggests that a protein functionally analogous to E. coli integration host factor may be present in H. influenzae.     

Intermolecular homologous recombination in plants.

To study DNA topological requirements for homologous recombination in plants, we have constructed pairs of plasmids that contain nonoverlapping deletions in the neomycin phosphotransferase gene [APH(3')II], which, when intact, confers kanamycin resistance to plant cells. Protoplasts isolated from Nicotiana tabacum were cotransformed with complementary pairs of plasmids containing these truncated gene constructs. Homologous recombination or gene conversion within the homologous sequences (6 to 405 base pairs) of the protein-coding region of the truncated genes led to the restoration of the functional APH(3')II gene, rendering these cells resistant to kanamycin. Circular plasmid DNAs recombined very inefficiently, independent of the length of the homologous region. A double-strand break in one molecule only slightly increased the recombination frequency. The most favorable substrates for recombination were linear molecules. In this case, the recombination frequency was positively correlated with the length of the homologous regions. The recombination frequency of plasmids linearized at sites proximal to the deletion-homology junction was significantly higher than when linearization was distal to the homologous region. Vector homology within cotransformed plasmid sequences also increased the recombination frequency.     

The recA-recBCD dependent recombination pathways of Serratia marcescens and Proteus mirabilis in Escherichia coli: functions of hybrid enzymes and hybrid pathways.

The physical maps of cloned recBCD gene regions of Serratia marcescens and Proteus mirabilis were correlated to genes located in this region. The genes thyA, recC, recB, recD and argA were organized as in Escherichia coli. The 3 rec genes code for the 3 different subunits of the RecBCD enzyme and produced enzymes promoting recombination and repair of UV damage in E coli. The recBCD-dependent stimulation of recombination at specific nucleotide sequences called Chi (Chi-activation) was determined in lambda red-gam-crosses. Chi-activation by the different RecBCD enzymes decreased in the order E coli greater than S marcescens greater than P mirabilis. In E coli cloned subunits genes from S marcescens and P mirabilis led to the formation of functional hybrid enzymes consisting of subunits from 2 or even 3 species. The origin of the RecC subunit present in the hybrid enzymes affected the degree of Chi-activation. Further, changes in Chi-activation occurred when the RecD subunit in the enzyme from E coli was replaced by RecD proteins from S marcescens or P mirabilis. This suggested that the RecD subunit determines not only whether or not Chi-activation is possible but also to which extent it occurs. Finally we have reconstituted recombination pathways of S marcescens and P mirabilis by combining the cloned recA and recBCD genes from these species in E coli deleted for recA and recBCD. Both pathways can efficiently promote recombination and repair. Studies are summarized which showed that levels of repair and recombination promoted by the recA-recBCD genes are mostly higher when the recA and recBCD genes came from the same species than from 2 different species (hybrid RecBCD recombination pathway). The data are interpreted to provide evidence that in vivo the RecA protein co-operates with the RecBCD enzyme in recombination and repair of UV damage.     

Homologous recombination and mutagenesis of gamma-irradiated plasmid DNA in Escherichia coli host cells

Plasmid DNA was used to study gamma-radiation-induced recombination and mutagenesis in Escherichia coli host cells. Plasmid pBRP1, a derivative of pBR322 containing the lac operon of E. coli, was irradiated with 60Co gamma rays prior to transformation into E. coli strains of different recA and lac genotypes. Plasmid-chromosome recombination was assayed in lacY1 host cells, whereas plasmid mutagenesis was assayed in delta lac host cells lacking chromosomal sequences homologous to the plasmid. Both recombinant and mutant plasmids were identified by the phenotypic changes in lactose utilization, and confirmed by restriction analysis of isolated plasmids. Plasmid-chromosome recombination was induced to high levels (about 20% of survivors at 700 Gy) and was dependent on the host recA gene. Plasmid mutagenesis occurred at lower levels (about 1.5% of survivors at 600 Gy) and was relatively independent of the recA gene. Plasmid survival was unaffected by the presence or absence of host recA mutations or the potential for plasmid-chromosome recombination.     

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.     

Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant

A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.     

Overcoming a defect in generalized recombination in the marine fish pathogen Vibrio anguillarum 775: construction of a recA mutant by marker exchange.

A defect in generalized recombination has prevented the use of marker exchange for the construction of specific chromosomal mutations in the marine fish pathogen Vibrio anguillarum 775. Through the use of large segments of homologous DNA, we were successful in overcoming this defect and used marker exchange to construct a recA mutant of V. anguillarum H775-3. A recombinant cosmid carrying the recA gene of V. anguillarum 775 in the center of a 25-kb cloned DNA insert was isolated by complementation of methyl methanesulfonate (MMS) sensitivity in Escherichia coli HB101. The recA gene was inactivated by inserting a kanamycin resistance gene into recA, and the mutant gene was subsequently introduced into V. anguillarum H775-3 by conjugal mobilization. Isolation of recombinants between cosmid-borne recA::kan sequences and chromosomal DNA was facilitated by the introduction of an incompatible plasmid, and Southern hybridization was used to verify the presence of recA::kan in the chromosomal DNA of the recA mutant. V. anguillarum carrying recA::kan was considerably more sensitive to UV radiation and to MMS than was its parent, and near wild-type levels of resistance to MMS and UV light were restored by introduction of cloned recA genes from both E. coli and V. anguillarum. These results indicate that recA is required for DNA repair in V. anguillarum and demonstrate the utility of this modified marker exchange technique for the construction of mutations in this economically important fish pathogen.     

An<Emphasis Type="Italic"> Arabidopsis</Emphasis> homologue of bacterial RecA that complements an<Emphasis Type="Italic"> E. coli recA</Emphasis> deletion is targeted to plant mitochondria

Homologous recombination results in the exchange and rearrangement of DNA, and thus generates genetic variation in living organisms. RecA is known to function in all bacteria as the central enzyme catalyzing strand transfer and has functional homologues in eukaryotes. Most of our knowledge of homologous recombination in eukaryotes is limited to processes in the nucleus. The mitochondrial genomes of higher plants contain repeated sequences that are known to undergo frequent rearrangements and recombination events. However, very little is known about the proteins involved or the biochemical mechanisms of DNA recombination in plant mitochondria. We provide here the first report of an Arabidopsis thaliana homologue of Escherichia coli RecA that is targeted to mitochondria. The mt recA gene has a putative mitochondrial presequence identified from the A. thaliana genome database. This nuclear gene encodes a predicted product that shows highest sequence homology to chloroplast RecA and RecA proteins from proteobacteria. When fused to the GFP coding sequence, the predicted presequence was able to target the fusion protein to isolated mitochondria but not to chloroplasts. The mitochondrion-specific localization of the mt recA gene product was confirmed by Western analysis using polyclonal antibodies raised against a synthetic peptide from a unique region of the mature mtRecA. The Arabidopsis mt recA gene partially complemented a recA deletion in E. coli, enhancing survival after exposure to DNA-damaging agents. These results suggest a possible role for mt recA in homologous recombination and/or repair in Arabidopsis mitochondria.     

Cloning of the Serratia marcescens recA gene and construction of a Serratia marcescens recA mutant

A recombinant plasmid, pSM2513, containing an 8.5 kb DNA insert was isolated from a genomic library of Serratia marcescens by using interspecific complementation. This plasmid conferred resistance to methyl methanesulphonate and UV irradiation upon recA mutants of Escherichia coli and enhanced recombination proficiency, as measured by Hfr-mediated conjugation, in recA mutants of E. coli. Furthermore, when recA mutants of E. coli harbouring pSM2513 were subjected to UV irradiation, filamentation of the cells was observed. This did not occur upon UV irradiation of the same mutants harbouring the cloning vector alone. These results imply that the S. marcescens recA gene on pSM2513 is functionally similar to the E. coli recA gene in several respects. Restriction enzyme analysis and subcloning studies revealed that the S. marcescens recA gene was located on a 2.7 kb Bg/II-KpnI fragment of pSM2513, and its gene product of approximately 39 kDa resembled the E. coli RecA protein in molecular mass. Using transformation-mediated marker rescue, a recA mutant of S. marcescens was successfully constructed; its proficiency both in homologous recombination and in DNA repair was abolished compared with its parent.     

Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30

Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.     

Molecular cloning and expression in Escherichia coli of the recA gene of Legionella pneumophila

Interspecific complementation of an Escherichia coli recA mutant with a Legionella pneumophila genomic library was used to identify a recombinant plasmid encoding the L. pneumophila recA gene. Recombinant E. coli strains harbouring the L. pneumophila recA gene were isolated by replica-plating bacterial colonies on medium containing methyl methanesulphonate (MMS). MMS-resistant clones were identified as encoding the L. pneumophila recA analogue by their ability to protect E. coli HB101 from UV exposure and promote homologous recombination. Subcloning of selected restriction fragments and Tn5 mutagenesis localized the recA gene to a 1.7 kb Bg/II-EcoRI fragment. Analysis of minicell preparations harbouring a 1.9 kb EcoRI fragment containing the recA coding segment revealed a single 37.5 kDa protein. Insertional inactivation of the cloned recA gene by Tn5 resulted in the disappearance of the 37.5 kDa protein, concomitant with the loss of RecA function. The L. pneumophila recA gene product did not promote induction of a lambda lysogen; instead, the presence of the heterologous recA gene caused a significant reduction in spontaneous and mitomycin-C-induced prophage induction in recA+ and recA E. coli backgrounds. Despite the lack of significant genetic homology between the L. pneumophila recA gene and the E. coli counterpart, the L. pneumophila RecA protein was nearly identical to that of E. coli in molecular mass, and the two proteins showed antigenic cross-reactivity. Western blot analysis of UV-treated L. pneumophila revealed a significant increase in RecA antigen in irradiated versus control cells, suggesting that the L. pneumophila recA gene is regulated in a manner similar to that of E. coli recA.