Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction

Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls into P22 particles. The transducing particle DNA contained duplications of the region of homology flanking the integrated plasmid vector sequences and lacked some phage genes. When these defective phage genomes containing the inserted plasmid infected a recipient cell, recombination between the duplicated regions regenerated the plasmid. A useful consequence of this sequence of events was that genetic markers in the region of homology were readily transferred from phage to plasmid. Plasmid transduction required homology between the phage and the plasmid, but did not depend on the presence of any specific P22 sequence in the plasmid. When the infecting P22 carried a DNA sequence homologous to the ampicillin resistance region of pBR322, the vector plasmid having no P22 insert could be transduced. P22-mediated transduction is a useful way to transfer chimeric plasmids, since most S. typhimurium strains are poorly transformed by plasmid DNA.     

Intergeneric conjugation in Streptomyces peucetius and Streptomyces sp. strain C5: chromosomal integration and expression of recombinant plasmids carrying the chiC gene

Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the phiC31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of phiC31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase.     

Mechanisms of spiroplasma genome variation associated with SpV1-like viral DNA inferred from sequence comparisons.

Genomes of Spiroplasma citri strains have rearranged frequently during their evolution, partly due to multiple integrated sequences of spiroplasma viruses. To understand better the role of viral sequences in genome evolution, we examined available nucleotide sequences of viruslike elements in the S. citri chromosome. Comparison of integrated and nonintegrated sequences of spiroplasma virus SpV1-C74 DNA suggested that it is an encapsidated form of the circular transposition intermediate belonging to an insertion sequence (IS3) family member. One SpV1-C74 viral DNA fragment was identified as interrupting the remains of a DNA adenine modification methylase gene. A viral DNA insertion of SpV1-R8A2 B DNA had hallmarks of having suffered an internal deletion by a site-specific recombination system. Homologous recombination likely was responsible for several deletions within viral DNA. A homologous recombination event was inferred between part of a viral DNA insertion and a similar chromosomal sequence. Dispersed sequences from SpV1-like C4 open reading frames (ORFs) were identified as involved in a complex deletion-inversion event. Thus, SpV1-like sequences likely have altered spiroplasma genomes by inserting within active genes, destroying their function, by providing targets for site-specific recombination, by mediating deletions of sequences adjacent to their integration sites, and by providing targets for homologous recombination, leading to inversions.     

Plasmid replication stimulates DNA recombination in Bacillus subtilis

The effects of plasmid replication on the frequency of homologous recombination have been investigated. For that purpose Bacillus subtilis strains that carry in their chromosome directly repeated DNA sequences, and an integrated copy of plasmid pE194 either proximal or distal to the repeats, were constructed. The repeat consists either of 3.9 X 10(3) base pBR322 sequences or 2.1 X 10(3) base B. subtilis chromosomal sequences. As plasmid pE194 is naturally thermosensitive for replication, the activity of the replicon could be regulated. Recombination between the repeated sequences was infrequent (about 10(-4) per generation) when the integrated plasmid did not replicate. It was 20 to 450 times higher when the plasmid was allowed to replicate, provided that the repeats were in the proximity of the plasmid. These results show that plasmid replication stimulates DNA recombination.     

Saccharomyces cerevisiae centromere CEN11 does not induce chromosome instability when integrated into the Aspergillus nidulans genome.

We constructed Aspergillus nidulans transformation plasmids containing the A. nidulans argB+ gene and either containing or lacking centromeric DNA from Saccharomyces cerevisiae chromosome XI (CEN11). The plasmids transformed an argB Aspergillus strain to arginine independence at indistinguishable frequencies. Stable haploid transformants were obtained with both plasmids, and strains were identified in which the plasmids had integrated into chromosome III by homologous recombination at the argB locus. Plasmid DNA was recovered from a transformant containing CEN11, and the sequence of the essential portion of CEN11 was determined to be unaltered. The transformants were further characterized by using them to construct heterozygous diploids and then testing the diploids for preferential loss of the plasmid-containing chromosomes. The CEN11 sequence had little or no effect on chromosome stability. Thus, CEN11 does not prevent chromosomal integration of plasmid DNA and probably lacks centromere activity in Aspergillus spp.     

Tn5-mediated transposition of plasmid DNA after transduction to Myxococcus xanthus.

After coliphage P1-mediated transfer of Tn5-containing plasmid DNA from Escherichia coli to Myxococcus xanthus, transductants were identified which contained plasmid sequences integrated at many sites on the bacterial chromosome. The unaltered plasmid DNA sequences in these transductants were apparently flanked by intact Tn5 or IS50 sequences. These results suggest that Tn5-mediated transposition has occurred and provide a method for integrating plasmid DNA into the M. xanthus chromosome without the requirement for homologous recombination.     

Recombinational instability of a chimeric plasmid in Saccharomyces cerevisiae.

Wild-type strains of Saccharomyces cerevisiae exhibit mitotic recombination between the chimeric plasmid TLC-1 and the endogenous 2mu circle that involves sequence homologies between the two plasmids that are not acted on by the 2mu circle site-specific recombination system. This generalized recombination can be detected because it separates the LEU2 and CAN1 markers of TLC-1 from each other through the formation of a plasmid containing only the S. cerevisiae LEU2 region and the 2mu circle. This derivative plasmid is maintained more stably during vegetative growth than TLC-1, and strains which carry it frequently lose the endogenous 2mu circle. Therefore, TLC-1 can provide a convenient selection for [cir0] cells. Formation of this new plasmid is greatly reduced, but not eliminated, in strains containing the rad52-1 mutation. This indicates that generalized mitotic recombination between plasmid sequences utilizes functions required for chromosomal recombination in S. cerevisiae.     

Recombination between the linear plasmid pPZG101 and the linear chromosome of Streptomyces rimosus can lead to exchange of ends

The 387 kb linear plasmid pPZG101 of Streptomyces rimosus R6 can integrate into the chromosome or form a prime plasmid carrying the oxytetracycline biosynthesis cluster. The integration of plasmid pPZG101 into the linear chromosome of S. rimosus R6-501 in mutant MV25 was shown to be due to a single cross-over at a 4 bp common sequence. pPZG101 had integrated into a 250 kb DNA sequence that was reiterated at a low level. This sequence includes the oxytetracycline biosynthesis cluster, so that homologous recombination generated a mixed population carrying different copy numbers of the region. The 1 Mb linear plasmid pPZG103 in mutant MV17 had also arisen from a cross-over between pPZG101 and the chromosome, so that one end of pPZG103 consists of c . 850 kb of chromosomal sequence including the oxytetracycline biosynthesis cluster. The plasmid pPZG101 was shown to consist of a unique central region of about 30 kb flanked by terminal inverted repeats of about 180 kb. Analysis of a presumed ancestor plasmid pPZG102 suggested that the long terminal repeats had arisen by a recombination event during the strain development programme.     

A bacterial model system for chromosomal targeting.

A system that permits efficient site-specific chromosomal targeting of foreign DNA on the Escherichia coli chromosome has been developed, using the FLP site-specific recombination system derived from the yeast 2 mu plasmid. The system demonstrates the feasibility of using site-specific recombination for this purpose, and provides a means to gather information on parameters that may affect chromosomal targeting to guide efforts to establish similar systems in higher eukaryotes. In this model system, the efficiency of integration of foreign DNA is affected by the location of the target site in the chromosome, and the structure of the recombination sites.     

Stability of the delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki in a recombinant strain of Clavibacter xyli subsp. cynodontis.

Deletion of chromosomally inserted gene sequences from Clavibacter xyli subsp. cynodontis, a xylem-inhabiting endophyte, was studied in vitro and in planta. We found that nonreplicating plasmid pCG610, which conferred resistance to kanamycin and tetracycline and contained segments of C. xyli subsp. cynodontis genomic DNA, integrated into a homologous sequence in the bacterial chromosome. In addition, pCG610 contains two copies of the gene encoding the CryIA(c) insecticidal protein of Bacillus thuringiensis subsp. kurstaki HD73. Using drug resistance phenotypes and specific DNA probes, we found that the loss of all three genes arose both in vitro under nonselective conditions and in planta. The resulting segregants are probably formed by recombination between the repeated DNA sequences flanking pCG610 that resulted from the integration event into the chromosome. Eventually, segregants predominated in the bacterial population. The loss of the integrated plasmid from C. xyli subsp. cynodontis revealed a possible approach for decreasing the environmental consequences of recombinant bacteria for agricultural use.     

Stability of the delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki in a recombinant strain of Clavibacter xyli subsp. cynodontis.

Deletion of chromosomally inserted gene sequences from Clavibacter xyli subsp. cynodontis, a xylem-inhabiting endophyte, was studied in vitro and in planta. We found that nonreplicating plasmid pCG610, which conferred resistance to kanamycin and tetracycline and contained segments of C. xyli subsp. cynodontis genomic DNA, integrated into a homologous sequence in the bacterial chromosome. In addition, pCG610 contains two copies of the gene encoding the CryIA(c) insecticidal protein of Bacillus thuringiensis subsp. kurstaki HD73. Using drug resistance phenotypes and specific DNA probes, we found that the loss of all three genes arose both in vitro under nonselective conditions and in planta. The resulting segregants are probably formed by recombination between the repeated DNA sequences flanking pCG610 that resulted from the integration event into the chromosome. Eventually, segregants predominated in the bacterial population. The loss of the integrated plasmid from C. xyli subsp. cynodontis revealed a possible approach for decreasing the environmental consequences of recombinant bacteria for agricultural use.     

Development of a cloning system in Mycoplasma pulmonis

A system suitable for recombinant DNA manipulation in mycoplasmas was developed using the cloned antibiotic-resistance genes of Tn4001 and Tn916. An integrative plasmid containing one of the resistance markers was inserted into the genome of Mycoplasma pulmonis to form a recipient strain. This was accomplished by transformation and homologous recombination between chromosomal DNA sequences cloned onto the integrative plasmid. A second vector, the cloning vector, containing the same plasmid replicon and alternate resistance marker, carried cloned foreign DNA. When transformed into mycoplasmal recipients, homologous recombination between plasmid sequences resulted in integration of the cloning vector and foreign DNA. A Brucella abortus gene coding for a 31-kDa protein and the P1 structural gene and operon from Mycoplasma pneumoniae were introduced to examine the feasibility of developing mycoplasma as cloning hosts. Recombinant plasmids as large as 20 kb were inserted into M. pulmonis, and the integrated foreign DNA was stably maintained. The maximum size of clonable DNA was not determined, but plasmids larger than 22 kb have not been transformed into mycoplasmas using polyethylene glycol. Also the size of genome (800-1200 kb) may affect the stability of larger inserts of foreign DNA. This system is applicable to any mycoplasma capable of transformation, homologous recombination and expression of these resistance markers. Because of their lack of a cell wall, mycoplasmas may be useful cloning hosts for membrane or excreted protein genes from other sources.     

Homology Search and Choice of Homologous Partner during Mitotic Recombination

Homologous recombination is an important DNA repair mechanism in vegetative cells. During the repair of double-strand breaks, genetic information is transferred between the interacting DNA sequences (gene conversion). This event is often accompanied by a reciprocal exchange between the homologous molecules, resulting in crossing over. The repair of DNA damage by homologous recombination with repeated sequences dispersed throughout the genome might result in chromosomal aberrations or in the inactivation of genes. It is therefore important to understand how the suitable homologous partner for recombination is chosen. We have developed a system in the yeast Saccharomyces cerevisiae that can monitor the fate of a chromosomal double-strand break without the need to select for recombinants. The broken chromosome is efficiently repaired by recombination with one of two potential partners located elsewhere in the genome. One of the partners has homology to the broken ends of the chromosome, whereas the other is homologous to sequences distant from the break. Surprisingly, a large proportion of the repair is carried out by recombination involving the sequences distant from the broken ends. This repair is very efficient, despite the fact that it requires the processing of a large chromosomal region flanking the break. Our results imply that the homology search involves extensive regions of the broken chromosome and is not carried out exclusively by sequences adjacent to the double-strand break. We show that the mechanism that governs the choice of homologous partners is affected by the length and sequence divergence of the interacting partners, as well as by mutations in the mismatch repair genes. We present a model to explain how the suitable homologous partner is chosen during recombinational repair. The model provides a mechanism that may guard the integrity of the genome by preventing recombination between dispersed repeated sequences.     

Inverted duplication of JH associated with chromosome 14 translocation and T-cell leukemia in ataxia-telangiectasia.

A specific 14q32 breakpoint is observed in a homologous chromosome 14 translocation [t(14;14)q12q32] occurring in the T-cells of about 10% of patients with ataxia-telangiectasia (AT). To investigate whether the 14q32 breakpoint in AT occurs within the immunoglobulin gene cluster as is frequently detected in B-cell lymphoma, immunoglobulin clones were hybridized to Southern blots of DNA isolated from the T-cells of two AT patients with this chromosome 14 translocation. The 14q32 translocation breakpoints in these patients are apparently not within JH, S mu, C mu, S alpha-1 or -2, or C alpha-1 or -2, but one of the patients has an inverted duplication of at least 26 kilobases (kb) of the C mu region, with an associated 5' flanking deletion. The point of origin of the inverted duplication is within JH near the recombination signal for the J4 gene. This suggests that normal JH recombination mechanisms may have played a role in the development of this 14q32 chromosomal aberration. The presence of AT chromosomal breakpoints near other rearranging genes suggests a role for exaggerated recombination in the pathogenesis of chromosomal instability in AT.     

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.     

The bacteriophage T4 insertion/substitution vector system. A method for introducing site-specific mutations into the virus chromosome

A bacteriophage T4 insertion/substitution vector system has been developed as a means of introducing in vitro generated mutations into the T4 chromosome. The insertion/substitution vector is a 2638-base pair plasmid containing the pBR322 origin of replication and ampicillin resistance determinant, a T4 gene 23 promoter/synthetic supF tRNA gene fusion, and a polylinker with eight unique restriction enzyme recognition sites. A T4 chromosomal "target" DNA sequence is cloned into this vector and mutated by standard recombinant DNA techniques. Escherichia coli cells containing this plasmid are then infected with T4 bacteriophage that carry amber mutations in two essential genes. The plasmid integrates into the T4 chromosome by recombination between the plasmid-borne T4 target sequence and its homologous chromosomal counterpart. The resulting phage, termed "integrants," are selectable by the supF-mediated suppression of their two amber mutations. Thus, although the integrants comprise 1-3% or less of the total phage progeny, growth on a nonsuppressing host permits their direct selection. The pure integrant phage can be either analyzed directly for a possible mutant phenotype or transferred to nonselective growth conditions. In the latter case, plasmid-free phage segregants rapidly accumulate due to homologous recombination between the duplicated target sequences surrounding the supF sequence in each integrant chromosome. A major fraction of these segregants will retain the in vitro generated mutation within their otherwise unchanged chromosomes and are isolated as stable mutant bacteriophage. The insertion/substitution vector system thereby allows any in vitro mutated gene to be readily substituted for its wild-type counterpart in the bacteriophage T4 genome.     

Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences.

To develop a method to modify genomic sequences in Ascobolus immersus by precisely reintroducing defined DNA segments previously manipulated in vitro, we investigated the effect of transforming DNA conformation on recombination with chromosomal sequences. Circular single-stranded DNA carrying the met2 gene and double-stranded DNA linearized by cutting within the met2 gene both transformed protoplasts of a met2 mutant strain of A. immersus to prototrophy. In contrast to the equivalent circular double-stranded DNA, which chiefly integrated at nonhomologous chromosomal sites, single-stranded and double-stranded cut DNAs recombined primarily with the homologous chromosomal met2 sequence. Of the single-stranded DNA transformants, 65% resulted from replacement of the resident met2 mutation by the exogenous wild-type allele. In 70% of the double-stranded-cut DNA transformants, one or more copies of the transforming DNA had integrated at the met2 locus, leading to tandem duplications of the met2 target region separated by plasmid DNA. These duplicated sequences could recombine, leading to progeny containing only one copy of the met2 region. This resulted in a precise gene replacement if the wild-type allele had been retained. In addition, we show that newly duplicated sequences were most often de novo methylated at the cytosine residues during the sexual phase. Cytosine methylation was associated with inactivation of the integrated met2 gene(s) in segregants of crosses. However, methylation was not accurately maintained at each DNA replication cycle, so that Met- segregants recovered a wild-type phenotype through successive mitotic divisions. This finding indicated that met2 genes were silenced by methylation alone.     

Suppression of the thermosensitive replication phenotype of the derivative plasmid of pI9789::Tn552 in Staphylococcus aureus may involve integration of the plasmid into the host chromosome

Abstract Plasmid-chromosome co-integration was found to be the mechanism of choice to overcome thermosensitivity of replication of the plasmid pS1 in PS80d and RN4220 strains of Staphylococcus aureus . The integration of the plasmid was sometimes accompanied by deletion of a specific section of the plasmid pS1 in PS80d. Growth of bacteriophage on strains containing the integrated plasmid and the subsequent use of the phage in transduction gave transductants containing plasmids that had regained their replication thermosensitivity. These plasmids had not acquired any detectable chromosomal DNA. The 16-kb EcoRI fragment of the PS80d chromosome that hybridizes to pS1 is the target for recombination in many cases, but apparently other sites are also used. This fragment contains sequence homologous to parts of the transposon Tn552 and it is probable that site-specific recombination is involved in the integration. The possible mechanisms for the integrations and the deletions are discussed.     

Coupled homologous and nonhomologous repair of a double-strand break preserves genomic integrity in mammalian cells

DNA double-strand breaks (DSBs) may be caused by normal metabolic processes or exogenous DNA damaging agents and can promote chromosomal rearrangements, including translocations, deletions, or chromosome loss. In mammalian cells, both homologous recombination and nonhomologous end joining (NHEJ) are important DSB repair pathways for the maintenance of genomic stability. Using a mouse embryonic stem cell system, we previously demonstrated that a DSB in one chromosome can be repaired by recombination with a homologous sequence on a heterologous chromosome, without any evidence of genome rearrangements (C. Richardson, M. E. Moynahan, and M. Jasin, Genes Dev., 12:3831-3842, 1998). To determine if genomic integrity would be compromised if homology were constrained, we have now examined interchromosomal recombination between truncated but overlapping gene sequences. Despite these constraints, recombinants were readily recovered when a DSB was introduced into one of the sequences. The overwhelming majority of recombinants showed no evidence of chromosomal rearrangements. Instead, events were initiated by homologous invasion of one chromosome end and completed by NHEJ to the other chromosome end, which remained highly preserved throughout the process. Thus, genomic integrity was maintained by a coupling of homologous and nonhomologous repair pathways. Interestingly, the recombination frequency, although not the structure of the recombinant repair products, was sensitive to the relative orientation of the gene sequences on the interacting chromosomes.