Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology

Summary To determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome. In this system the recombination frequencies were measured between is pE194 derivatives carrying segments of the chromosomal -gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature. Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for -gluconase activity. A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp. It was found that approximately 70 bp of homology is required for detectable homologous recombination. Homologous recombination was not detected when only 25 by of homology between plasmid and chromosome were provided. The data indicate that homology requirements for recombination in B. subtilis differ from those in Escherichia coli.     

The RecE recombination pathway mediates recombination between partially homologous DNA sequences: structural analysis of recombination products

Escherichia coli generalized recombination, utilizing the RecA RecB recombination pathway, requires large stretches (70-200 bp) of complete DNA sequence homology. In contrast, we have found that the RecE pathway can promote recombination between DNA with only short stretches of homology. A plasmid containing 10 partially homologous direct repeats was linearized by digestion with specific restriction enzymes. After transformation, a RecE+ (sbcA) host was able to circularize the plasmid by recombination between partially homologous direct repeat sequences. Recombination occurred in regions of as little as 6 bp of perfect homology. Recombination was enhanced in the regions adjacent to restriction sites used to linearize the plasmid, consistent with a role of double-strand breaks in promoting recombination. A mechanism is proposed in which the 5' exonuclease, ExoVIII, produces 3' single-stranded ends from the linearized plasmid. These pair with other sequences of partial homology. Partial homologies in the sequences flanking the actual join serve to stabilize this recombination intermediate. Recombination is completed by a process of "copy and join." This recombination mechanism requires less homology to stabilize intermediates than the degree of homology needed for mechanisms involving strand invasion. Its role in nature may be to increase genomic diversity, for example, by enhancing recombination between bacteriophages and regions of the bacterial chromosome.     

An efficient method of constructing homologous recom binant baculovirus with PCR-amplified fragments

Baculovirus is a rod-shaped virus containing a large circular dsDNA genome with the size of 80—180 kb[1]. Baculoviruses have been used as insecticides for biological control of forest and agricultural pests[2]. In addition, baculovirus is of great interest as it can be used as efficient eukaryotic expression vector[3], surface display vector[4], and gene therapy vector[5]. Till April 2002, the complete genome sequences of 13 baculoviruses have been reported. The functional genomics has now…     

Theta-type DNA replication stimulates homologous recombination in the Bacillus subtilis chromosome

To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of ∼ 4 kb. The replicon was derived from a broad-host-range plasmid, pAMβ1, which replicates by a unidirectional theta mechanism and is thermosensitive. The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194. Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats. The integrated replicon was active at the permissive temperature, and ∼ 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin. Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats. We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.     

Construction of plasmids integrating into the Bacillus subtilis chromosome through homologous recombination and their use as integration vectors

We constructed a number of plasmids which integrate into the chromosome of Bacillus subtilis through homology recombination. Plasmids consist of pBR322 replicon, different fragments of Bac. subtilis chromosomal DNA, Cm resistance marker from pBD64 plasmid. Frequency of transformation was 10(-4) per bacterial cell. Foreign DNA (genes for tryptophan metabolism of Bac. mesentericus) was introduced into the chromosome of Bac. subtilis with the help of these plasmids.     

Identification of plasmid and Bacillus subtilis chromosomal recombination sites used for pE194 integration.

The plasmid pE194 (3.7 kilobases) is capable of integrating into the genome of the bacterial host Bacillus subtilis in the absence of the major homology-dependent RecE recombination system. Multiple recombination sites have been identified on both the B. subtilis chromosome and pE194 (J. Hofemeister, M. Israeli-Reches, and D. Dubnau, Mol. Gen. Genet. 189:58-68, 1983). The B. subtilis chromosomal recombination sites were recovered by genetic cloning, and these sites were studied by nucleotide sequence analysis. Recombination had occurred between regions of short nucleotide homology (6 to 14 base pairs) as indicated by comparison of the plasmid and the host chromosome recombination sites with the crossover sites of the integration products. Recombination between the homologous sequences of the plasmid and the B. subtilis genome produced an integrated pE194 molecule which was bounded by direct repeats of the short homology. These results suggest a recombination model involving a conservative, reciprocal strand exchange between the two recombination sites. A preferred plasmid recombination site was found to occur within a 70-base-pair region which contains a GC-rich dyad symmetry element. Five of seven pE194-integrated strains analyzed had been produced by recombination at different locations within this 70-base-pair interval, located between positions 860 and 930 in pE194. On the basis of these data, mechanisms are discussed to explain the recombinational integration of pE194.     

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.     

An efficient method of constructing homologous recom binant baculovirus with PCR-amplified fragments

This paper describes a rapid method of constructing homologous recombinant baculovirus inE. coli with PCR-amplified fragments. By using this method, the traditional steps of constructing transfer vector are omitted. The method is based on phage λ red system which can promote the recombination between the homologous fragments with the length above 36 bp. Taking HaSNPV as an example, this paper describes the rapid recombination process by using chloramphenicol resistance gene (Cm ^R ) to replaceorf135 in HaSNPV genome. A pair of primers with length of 60 bp was synthesized, in which 40 bp was homologous to the each end sequence oforf135, and the rest 20 bp was homologous to the each end sequence ofCm ^R . By using these primers, a linear fragment containing the completeCm ^R gene between 40 bp of homologous arms oforf135 was generated by PCR with the plasmid pKD3 which containsCm ^R as the template. By transforming the linear fragment into theE. coli containing the bacterial artificial chromosome of HaSNPV and with the help of a plasmid expressing λ recombinase, the recombinants on which the homologue replacement had taken place were selected by chloramphenicol resistance. This method greatly shortens the process of constructing recombinant baculovirus since the process was performed inE. coli and does not need to construct transfer vectors. It can be further used for gene replacement and gene deletion of other large viral genomes.     

Recombination of DNAs in Xenopus oocytes based on short homologous overlaps.

Linear molecules of pBR322 and closely related plasmid DNAs were injected into Xenopus oocyte nuclei. Such molecules were degraded unless their ends were recombined. Non-homologous ends were joined rarely, if at all, but measurable recombination was supported by homologous sequences of less than 10 base pairs (bp). The efficiency of recombination increased as the length and degree of homology improved, in the range of about 8-20 bp. The homologous sequences had to be very close to the original molecular ends (within about 20 bp); internal homologies, even when they included better matches, were never used. These observations are best accommodated by a model of recombination which envisions exonucleolytic resection to expose homologous sequences, followed by annealing of single-stranded tails, tidying up and sealing of the new joint. Some of the recombined plasmids had novel tetracycline resistance genes; their properties give some insight into the function of the tet gene product.     

Intermolecular recombination during transformation of Bacillus subtilis competent cells by monomeric and dimeric plasmids

Bacillus subtilis competent cells harboring plasmid pUB110 were transformed by plasmids unable to replicate in this host but carrying segments of pUB110, 260 to 4500 bp long. Recombinants between the incoming and the resident plasmids were found in the transformed cells. Transforming efficiency of the incoming plasmids depended strongly on their molecular form and the length of their region homologous with the resident plasmid. It increased with the fourth to fifth power of that length for monomers having at least 900 bp of homology. Activity of monomers having less than 900 bp homology was too low to be measured in our experiments. Transforming efficiency of dimers was much greater than that of monomers, and varied with the square of the length of the homologous region. These results indicate that dimeric and monomeric plasmid molecules are processed differently during transformation of B. subtilis competent cells.     

Induction of recombination between homologous and diverged DNAs by double-strand gaps and breaks and role of mismatch repair.

Sequence homology is expected to influence recombination. To further understand mechanisms of recombination and the impact of reduced homology, we examined recombination during transformation between plasmid-borne DNA flanking a double-strand break (DSB) or gap and its chromosomal homolog. Previous reports have concentrated on spontaneous recombination or initiation by undefined lesions. Sequence divergence of approximately 16% reduced transformation frequencies by at least 10-fold. Gene conversion patterns associated with double-strand gap repair of episomal plasmids or with plasmid integration were analyzed by restriction endonuclease mapping and DNA sequencing. For episomal plasmids carrying homeologous DNA, at least one input end was always preserved beyond 10 bp, whereas for plasmids carrying homologous DNA, both input ends were converted beyond 80 bp in 60% of the transformants. The system allowed the recovery of transformants carrying mixtures of recombinant molecules that might arise if heteroduplex DNA--a presumed recombination intermediate--escapes mismatch repair. Gene conversion involving homologous DNAs frequently involved DNA mismatch repair, directed to a broken strand. A mutation in the PMS1 mismatch repair gene significantly increased the fraction of transformants carrying a mixture of plasmids for homologous DNAs, indicating that PMS1 can participate in DSB-initiated recombination. Since nearly all transformants involving homeologous DNAs carried a single recombinant plasmid in both Pms+ and Pms- strains, stable heteroduplex DNA appears less likely than for homologous DNAs. Regardless of homology, gene conversion does not appear to occur by nucleolytic expansion of a DSB to a gap prior to recombination. The results with homeologous DNAs are consistent with a recombinational repair model that we propose does not require the formation of stable heteroduplex DNA but instead involves other homology-dependent interactions that allow recombination-dependent DNA synthesis.     

recE4-Independent recombination between homologous deoxyribonucleic acid segments of Bacillus subtilis plasmids.

A plasmid (pLS104) carrying a tandem repetition of the leu region of the Bacillus subtilis chromosome arose spontaneously from pLS103, which carried a single copy of the leu region. Plasmid preparations from strains harboring pLS104 also contained the original plasmid, pLS103, and, in some preparations, plasmids carrying three or four repetitions of the leu region. These plasmids were shown to be generated by recombination between homologous deoxyribonucleic acid (DNA) segments in the tandemly repeated DNA regions on the plasmids, but not by recombinations between specific DNA sites. These phenomena were observed in a recE4-Independent background, showing that recombination of the homologous DNA sequences does not require the recE-Independent gene product(s).     

Integration ofhup cosmid pHU52 into the chromosomal DNA ofCicer-Rhizobium using Tn5 as an homologous sequence

Cosmid pHU52, which carrieshup genes ofBradyrhizobium japonicum, has been integrated into theCicer-Rhizobium G36-84 genome via Tn5-mediated homologous recombination. Tn5 was inserted into both the cosmid pHU52 and the chromosome ofCicer-Rhizobium to provide a region of DNA homology, without affecting the expression of necessary genes. An incompatible plasmid, pPH1JI, was used to select those few cells that had undergone recombination. The integration of the cosmid was demonstrated by Southern blot analysis. Chromosomal integration of thehup genes maximized stability and minimized the potential for their horizontal transfer to other bacterial species. The integratedhup genes were found to expressex planta as well in nodules. The method described illustrates how a given gene can be stably integrated into the chromosome.     

Characterization of RAD51-independent break-induced replication that acts preferentially with short homologous sequences

Repair of double-strand breaks by gene conversions between homologous sequences located on different Saccharomyces cerevisiae chromosomes or plasmids requires RAD51. When repair occurs between inverted repeats of the same plasmid, both RAD51-dependent and RAD51-independent repairs are found. Completion of RAD51-independent plasmid repair events requires RAD52, RAD50, RAD59, TID1 (RDH54), and SRS2 and appears to involve break-induced replication coupled to single-strand annealing. Surprisingly, RAD51-independent recombination requires much less homology (30 bp) for strand invasion than does RAD51-dependent repair (approximately 100 bp); in fact, the presence of Rad51p impairs recombination with short homology. The differences between the RAD51- and RAD50/RAD59-dependent pathways account for the distinct ways that two different recombination processes maintain yeast telomeres in the absence of telomerase.     

Distribution of bacteriophage phi 3T homologous deoxyribonucleic acid sequences in Bacillus subtilis 168, related bacteriophages, and other Bacillus species.

The Bacillus subtilis 168 chromosome was found to share extensive homology with the genome of bacteriophage phi 3T. At least three different regions of the bacterial genome hydridized to ribonucleic acid complementary to phi 3T deoxyribonucleic acid (DNA). The thymidylate synthetase gene, thyA, of B. subtilis and the sequences adjacent to it were shown to be homologous to the region in the phi 3T DNA containing the phage-encoded thymidylate synthetase gene, thyP3. SP beta, a temperate bacteriophage known to be integrated into the B. subtilis 168 chromosome, was demonstrated to be closely related to phi 3T. Other regions of the bacterial genome were also found to hybridize to the phi 3T probe. The nature and location of these sequences in the bacterial and phage chromosomes were not identified. It was shown however, that they were not homologous to either the thyP3 gene or the DNA surrounding the thyP3 gene. The chromosomes of other Bacillus species were also screened for the presence of phi 3T homologous sequences, and the thyP3 gene was localized in the linear genomes of phages phi 3T and rho 11 by heteroduplex mapping. It is suggested that the presence of sequences of phage origin in the B. subtilis 168 chromosome might contribute to the restructuring and evolution of the viral and bacterial DNAs.     

Integration of heterologous plasmid DNA into multiple sites on the genome of Campylobacter coli following natural transformation.

The efficiency of homologous recombination in Campylobacter coli following the introduction of DNA by natural transformation was determined by using a series of nonreplicating integrative vectors containing DNA fragments derived from the C. coli catalase gene. Homologous recombination occurred with as little as 286 homologous bp present and was not detected when 270 bases of homology was provided. Instead, when plasmids with little or no homology to the chromosome were introduced by natural transformation, the vector DNA became chromosomally integrated at random sites scattered throughout the C. coli genome. Southern analysis and nucleotide sequencing revealed that recombination had occurred between nonhomologous sequences and can therefore be described as illegitimate. There were at least five different recombination sites on plasmid pSP105. The ability of C. coli to acquire heterologous plasmids by natural transformation, and maintain them by chromosomal integration following illegitimate recombination, has fascinating implications for the genomic diversity and evolution of this species.     

Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions.

We present an intermolecular recombination assay for mammalian cells that does not involve the reconstitution of a selectable marker. It is based on the generation of a shuttle vector by recombination between a bacterial and a mammalian vector. The recombinants can thus be amplified in mammalian cells, isolated by plasmid rescue in an Escherichia coli RecA- host, and identified by in situ hybridization, by using mammalian vector sequences as probes. Since both parental molecules can share defined lengths of homology, this assay permits a direct comparison between homologous and nonhomologous intermolecular recombination. Our results indicate that the dominant intermolecular recombination mechanism is a nonhomologous one. The relative frequency of homologous to nonhomologous recombination was influenced by the length of shared homology between parental molecules and the replicative state of the parental molecules, but not by the introduction of double-strand breaks per se. Finally, almost all of the recombinants with a homologous junction did not have the reciprocal homologous junction but instead had a nonhomologous one. We propose a model to account for the generation of these recombinants.     

Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata

The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination.     

Requirements for ectopic homologous recombination in mammalian somatic cells.

Ectopic recombination occurs between DNA sequences that are not in equivalent positions on homologous chromosomes and has beneficial as well as potentially deleterious consequences for the eukaryotic genome. In the present study, we have examined ectopic recombination in mammalian somatic (murine hybridoma) cells in which a deletion in the mu gene constant (Cmu) region of the endogenous chromosomal immunoglobulin mu gene is corrected by using as a donor an ectopic wild-type Cmu region. Ectopic recombination restores normal immunoglobulin M production in hybridomas. We show that (i) chromosomal mu gene deletions of 600 bp and 4 kb are corrected less efficiently than a deletion of only 2 bp, (ii) the minimum amount of homology required to mediate ectopic recombination is between 1.9 and 4.3 kb, (iii) the frequency of ectopic recombination does not depend on donor copy number, and (iv) the frequency of ectopic recombination in hybridoma lines in which the donor and recipient Cmu regions are physically connected to each other on the same chromosome can be as much as 4 orders of magnitude higher than it is for the same sequences located on homologous or nonhomologous chromosomes. The results are discussed in terms of a model for ectopic recombination in mammalian somatic cells in which the scanning mechanism that is used to locate a homologous partner operates preferentially in cis.     

High-efficiency gene inactivation and replacement system for gram-positive bacteria.

A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37 degrees C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37 degrees C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 degrees C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.