Characterization of eight excision plasmids of Pseudomonas syringae pv. phaseolicola

Summary Pseudomonas syringae pv. phaseolicola strain LR719 contains a 150 kilobase pair (kb) plasmid pMC7105, stably integrated into its chromosome. Occasionally, single colony isolates of this strain contain an excision plasmid. Eight unique excision plasmids were selected and characterized by BamHI restriction endonuclease and blot hybridization analyses. These plasmids ranged in size from 35 to 270 kb; the largest contained approximately 130 kb of chromosomal DNA sequences. Restriction maps of pMC7105 were developed to deduce the site of integration and to identify the fragments in which recombination occurred to produce each excision plasmid. The eight excision plasmids were arranged into five classes based on the sites where excision occurs. A 20 kb region of pMC7105, which includes BamHI fragment 9 and portions of adjacent fragments, is present in all excision plasmids and thought to contain the origin of replication. The site of integration on pMC7105 maps within BamHI fragment 8. This fragment shows homology with seven other BamHI fragments of pMC7105 and with five chromosomal fragments identified among the excision plasmids. The data strongly suggest that the integration of pMC7105 may have occurred at a repetitive sequence present on the chromosome and on the plasmid.     

Integration and partial excision of a cryptic plasmid in Pseudomonas syringae pv. phaseolicola.

A virulent strain of Pseudomonas syringae pv. phaseolicola, a pathogen of the common bean Phaseolus vulgaris (L.), was shown to harbor a 98-megadalton cryptic plasmid, pMC7105. After exposure of this strain to the plasmid-curing agent mitomycin C, a colony was isolated which had no detectable extrachromosomal DNA. Hybridization of labeled pMC7105 probe to nitrocellulose filters containing Southern-blotted BamHI cleavage products of cellular DNA revealed that pMC7105 was integrated into the chromosome rather than cured from this strain. Imprecise excision of pMC7105 resulted in the formation of three smaller plasmids of 34, 50, and 58 megadaltons. BamHI and EcoRI fingerprint analyses revealed that these plasmids were excised from a common region of pMC7105. The BamHI fragments of pMC7105 which were not present in the excision plasmids remained integrated and could be detected by hybridization of pMC7105 probe to Southern-blotted cellular DNA from these strains. Certain chromosomal fragments also had homology with the pMC7105 probe. The excision plasmids were stably maintained and neither integration nor excision altered the pathogenicity of these strains.     

Identification and mapping of regions that confer plasmid functions and of sites for excisive recombination of plasmid pMMC7105

Summary Strain PP808 of Pseudomonas syringae pv. phaseolicola contains pEXC8080 (34.6 kb), the smallest of several plasmids that originated by partial excision of the cryptic plasmid, pMMC7105 (150 kb), from the host chromosome. This excision plasmid is derived entirely of sequences from pMMC7105 and contains a 24 kb region referred to as common DNA, which is present in each of the other excision plasmids. A six enzyme restriction endonuclease map was constructed of pEXC8080. The replication region was mapped by identifying small restriction fragments that conferred replication properties to pMB1 plasmids that otherwise fail to replicate in Pseudomonas. This region is located within the common DNA and is 0.8–3.8 kb in size. Sequences from pEXC8080 failed to stabilize pMB1 derivatives in Pseudomonas in the absence of antibiotic selection, but stability functions were mapped to a region of pMMC7105 that presumably remains integrated in the chromosome of strain PP808. An incompatibility region was mapped to a 7.3 kb region on pEXC8080 that is closely linked to, but not included within, the replication region. The recombination site was mapped to a 1.2 kb region of the fusion fragment that was formed upon excision of pEXC8080. RS-I, a repetitive sequence, found on pMMC7105 was present in the fusion fragment at the site of recombination. RS-I was also mapped to BamHI fragments that recombined upon excision of pEXC8080 and suggest that it provides sites for homologous recombination.     

An integrative plasmid and multiple-sized plasmids of Pseudomonas syringae pv. phaseolicola have extensive homology

Summary Plasmids isolated from five strains of the bean pathogen Pseudomonas syringae pv. phaseolicola were characterized by restriction endonuclease and filter hybridization analyses. BamHI and EcoRI restriction patterns revealed that total plasmid DNA from each strain had a high level of sequence homology with pMC7105, a 148 kbp integrative plasmid found in a sixth strain. Only six BamHI fragments from the eight plasmids in these strains failed to hybridize with pMC7105 probe. Four of these fragments, three from pPP6520 and one from pPP6525 of strain PP652, hybridized strongly to plasmid DNA from a closely-related pathovar, P. syringae pv. glycinea. BamHI fragment 8, which is involved in the integration of pMC7105 into the host chromosome, contains a repeat sequence that was present on all the plasmids except pPP6120 (6.8 kbp), pPP6310 (40 kbp) and pPP6520 (45 kbp). Every plasmid but pPP6520 had fragments that showed weak hybridization to the small plasmid, pPP6120. This homology suggests that a second repetitive sequence is common to these plasmids. The large plasmids (148 to 151 kbp) were essentially identical to pMC7105. The intermediate plasmids (122 to 128 kbp) appeared to be derived mainly from pMC7105 or a related plasmid, whereas the smaller plasmids (6.8 to 45 kbp) appear to have been derived in part from sequences not present in pMC7105.     

The integrated and free states of Streptomyces griseus plasmid pSG1

A 16.6-kb plasmid-pSG1-was isolated from Streptomyces griseus following transformation of protoplasts with unrelated plasmids. Southern hybridization experiments with radioactive probes prepared from pSG1 fragments and immobilized S. griseus DNA fragments indicated that the plasmid was present in the progenitor strain, in an integrated state. In the pSG1+ isolates plasmid sequences existed both as integrated sequences and as free plasmids. The integrated state of maintenance persisted in strains which have been cured of the free plasmid. The junction site on the plasmid was located on a 0.5-kb EcoRI-SalI fragment. The chromosomal integration site was demonstrated to be the same in all strains derived from S. griseus NRRL3851. The occurrence of both states of plasmid maintenance in the same clones indicates that an integrated pSG1 sequence does not interfere with free plasmid replication and partition. It suggests that the establishment of the free state may involve a replicative excision of pSG1 from the S. griseus chromosome.     

Molecular cloning of eucaryotic genes required for excision repair of UV-irradiated DNA: isolation and partial characterization of the RAD3 gene of Saccharomyces cerevisiae.

We describe the molecular cloning of a 6-kilobase (kb) fragment of yeast chromosomal DNA containing the RAD3 gene of Saccharomyces cerevisiae. When present in the autonomously replicating yeast cloning vector YEp24, this fragment transformed two different UV-sensitive, excision repair-defective rad3 mutants of S. cerevisiae to UV resistance. The same result was obtained with a variety of other plasmids containing a 4.5-kb subclone of the 6-kb fragment. The UV sensitivity of mutants defective in the RAD1, RAD2, RAD4, and RAD14 loci was not affected by transformation with these plasmids. The 4.5-kb fragment was subcloned into the integrating yeast vector YIp5, and the resultant plasmid was used to transform the rad3-1 mutant to UV resistance. Both genetic and physical studies showed that this plasmid integrated by homologous recombination into the rad3 site uniquely. We conclude from these studies that the cloned DNA that transforms the rad3-1 mutant to UV resistance contains the yeast chromosomal RAD3 gene. The 4.5-kb fragment was mapped by restriction analysis, and studies on some of the subclones generated from this fragment indicate that the RAD3 gene is at least 1.5 kb in size.     

A specialized host-vector system for the in Vivo cloning of the trp operon of wild-type and mutant strains of Salmonella typhimurium by generalized transduction

Using in vitro methods, a 14.2-kb EcoRI fragment of the Salmonella typhimurium chromosome containing the trp operon plus associated flanking sequences from deletion mutant delta trpDCB763 was cloned into the EcoRI site of plasmid pBR322 in a S. typhimurium host. An in vivo cloning vector was constructed from the recombinant plasmid by the in vitro excision of a SalI fragment that contains the entire trp operon. The derived plasmid (pSTP21) carries a hybrid insert made up of the 5.4-kb EcoRI-SalI upstream flanking sequence and the 3.2-kb SalI-EcoRI downstream flanking sequence. Plasmid pSTP21 has been used as a receptor plasmid to clone a variety of mutant and wild-type trp operons by RecA-dependent in vivo recombination between the insert DNA of the plasmid and the homologous trp flanking sequences of transducing DNA fragments transferred into the cell by bacteriophage P22. The host-vector system developed for the in vivo cloning permits the differentiation of plasmid transductants from chromosomal transductants on the primary selective medium. Expression of the cloned trp operons is regulated normally by tryptophan. A substantial amplification of trp enzymes is attainable upon derepression. The recombinant plasmids are stably inherited in RecA+ and RecA- S. typhimurium hosts. However, conditions of high expression of the trp operon lead to a rapid loss of cellular viability and of plasmid stability.     

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.     

Homology of pCS1 Plasmid Sequences with Chromosomal DNA in Clavibacter michiganense subsp. sepedonicum: Evidence for the Presence of a Repeated Sequence and Plasmid Integration

Restriction fragments of pCS1, a 50.6-kilobase (kb) plasmid present in many strains of Clavibacter michiganense subsp. sepedonicum (“Corynebacterium sepedonicum”), have been cloned in an M13mp11 phage vector. Radiolabeled forms of these cloned fragments have been used as Southern hybridization probes for the presence of plasmid sequences in chromosomal DNA of this organism. These studies have shown that all tested strains lacking the covalently closed circular form of pCS1 contain the plasmid in integrated form. In each case the site of integration exists on a single plasmid restriction fragment with a size of 5.1 kb. Southern hybridizations with these probes have also revealed the existence of a major repeated sequence in C. michiganense subsp. sepedonicum. Hybridizations of chromosomal DNA with deletion subclones of a 2.9-kb plasmid fragment containing the repeated sequence indicate that the size of the repeated sequence is approximately 1.3 kb. One of the copies of the repeated sequence is on the plasmid fragment containing the site of integration.     

Use of a transposon-encoded site-specific resolution system for construction of large and defined deletion mutations in bacterial chromosome

A class II transposon, Tn1722, encodes a site-specific resolution system, in which the resolvase (TnpR) efficiently catalyzes intramolecular recombination between the two directly oriented copies of the resolution site (res), leading to precise excision of the intervening DNA region. This property was exploited to develop the general strategies to introduce the large and defined deletion mutations into the bacterial chromosome. The Tn1722 res site was inserted into the plasmid carrying a cloned chromosomal fragment, and the resulting plasmid was integrated into a Tn1722-containing target chromosome by single crossover-mediated homologous recombination. The plasmid integrant carrying the two copies of the res site in the same orientation could efficiently excise the chromosomal region locating between the two res sites by means of the site-specific resolution system. Such site-specific deletion could be also detected by appropriate integration of the res–tnpR-containing plasmid into the chromosome in which another copy of the res site had been inserted through allelic exchange. This latter strategy was further modified to isolate the deletion mutations that were free of the resistance markers used for introduction of the res site and the res–tnpR block into the target chromosome. The deletion systems were applied to analyze the 103-kb pvd region of Pseudomonas aeruginosa PAO carrying most of the pyoverdin biosynthetic genes. Successful isolation of the mutation lacking more than a 100-kb fragment in the pvd region indicated that this region did not carry any essential genes.     

Construction of a Bacillus subtilis cloning vehicle with heterologous DNA sequence

A cloning vehicle, pFTB91, for the Bacillus subtilis host was constructed with DNA fragments heterologous to the host chromosome. It consists of three DNA fragments: (i) chromosomal DNA of Bacillus amyloliquefaciens which complements the leuA and ilvC mutations in B. subtilis; (ii) a B. amyloliquefaciens plasmid DNA that supplies an autonomously replicating function; and (iii) a HindIII fragment of Staphylococcus aureus plasmid pTP5 that carries gene tetr, conferring the TetR phenotype. It has sufficiently low DNA homology to prevent its integration into the host chromosome in recombination-competent cells of B. subtilis. It is 9.3 kb, and approx. 10 copies are present per chromosome. The SalI and KpnI sites in the ilvC+ and tetr genes, respectively, could be used for selection of recombinant plasmids by insertional inactivation. The plasmid has unique sites for EcoRI, PstI, and XbaI.     

Genetic and physical characterization of recombinant plasmids associated with cell aggregation and high-frequency conjugal transfer in Streptococcus lactis ML3.

Restriction mapping was employed to characterize the 104-kilobase (kb) cointegrate lactose plasmids from 15 independent transconjugants derived from Streptococcus lactis ML3 as well as the 55-kb lactose plasmid ( pSK08 ) and a previously uncharacterized 48.4-kb plasmid ( pRS01 ) from S. lactis ML3. The data revealed that the 104-kb plasmids were cointegrates of pSK08 and pRS01 and were structurally distinct. The replicon fusion event occurred within adjacent 13.8- or 7.3-kb PvuII fragments of pSK08 and interrupted apparently random regions of pRS01 . Correlation of the transconjugants' clumping and conjugal transfer capabilities with the interrupted region of pRS01 identified pRS01 regions coding for these properties. In the 104-kb plasmids, the pRS01 region was present in both orientations with respect to the pSK08 region. The replicon fusion occurred in recombination-deficient (Rec-) strains and appeared to introduce a 0.8 to 1.0-kb segment of DNA within the junction fragments. The degeneration of the cointegrate plasmids was monitored by examining the lactose plasmids from nonclumping derivatives of clumping transconjugants. These plasmids displayed either precise or imprecise excision of pRS01 sequences or had dramatically reduced copy numbers. Both alterations occurred by rec-independent mechanisms. Alterations of a transconjugant 's clumping phenotype also occurred by rec-independent inversion of a 4.3-kb KpnI-PvuII fragment within the pRS01 sequences of the cointegrate plasmid.     

Dynamic plasmid populations in Halobacterium halobium.

Deletion events occurring in the major 150-kilobase-pair (kb) plasmid pHH1 of the archaebacterium Halobacterium halobium were investigated. We found four deletion derivatives of pHH1 in gas-vacuole-negative mutants, two of which (pHH23) [65 kb] and pHH4 [36 kb]) we analyzed. Both plasmids incurred more than one deletion, leading to the fusion of noncontiguous pHH1 sequences. pHH23 and pHH4 overlapped by only 4 kb of DNA sequence. A DNA fragment derived from this region was used to monitor the production of further deletion variants of pHH4. A total of 25 single colonies were characterized, 23 of which contained various smaller pHH4 derivatives. Of the 25 colonies investigated, 2 had lost pHH4 entirely and contained only large (greater than or equal to 100-kb) minor covalently closed circular DNAs. One colony contained the 17-kb deletion derivative pHH6 without any residual pHH4. The sizes of the pHH4 deletion derivatives, produced during the development of a single colony, ranged from 5 to 20 kb. In five colonies, pHH4 was altered by the integration of an additional insertion element. These insertions, as well as copies of the various insertion elements already present in pHH4, presumably serve as hot spots for recombination events which result in deletions. A second enrichment procedure led to the identification of colonies containing either a 16-kb (pHH7) or a 5-kb (pHH8) deletion derivative of pHH4 as the major plasmid. pHH8, the smallest plasmid found, contained the 4 kb of unique DNA sequence shared by pHH23 and pHH4, as well as some flanking pHH4 sequences. This result indicates that the 4-kb region contains the necessary sequences for plasmid maintenance and replication.     

Construction of isogenic gonococcal strains varying in the presence of a 4.2-kilobase cryptic plasmid.

A 4.2-kilobase (kb) cryptic plasmid is present in 96% of isolates of Neisseria gonorrhoeae. An inability to construct isogenic derivatives which vary in the presence of the 4.2-kb plasmid has prevented the study of its function. We report a method to deliver an intact 4.2-kb plasmid into plasmidless gonococcal strains. The method involved transformation with novel 15.7-kb hybrid penicillinase-producing (Pcr) plasmids, which were cointegrates containing two copies of the 4.2-kb plasmid arranged in tandem direct repeat plus one copy of the 7.2-kb Pcr plasmid pFA3. When the 15.7-kb hybrid Pcr plasmids were introduced into a gonococcal recipient lacking evident plasmids, they dissociated at a relatively high frequency into plasmids identical to their parents: the 4.2-kb cryptic plasmid and pFA10 (a stable 11.5-kb plasmid containing one copy of each of the 7.2-kb Pcr plasmid pFA3 and the 4.2-kb cryptic plasmid pFA1). Curing strains of their Pcr plasmids resulted in isogenic strains which varied only in the presence of the 4.2-kb plasmid. The presence of the autonomously replicating 4.2-kb plasmid did not affect a number of tested phenotypes, including auxotype, antibiotic sensitivity, and frequencies of variation of outer membrane protein II. The interpretation of the functional significance of the 4.2-kb plasmid was complicated, however, by the additional finding that each of three tested plasmid-free strains contained a chromosomal fragment of about 1.6 kb that hybridized under moderate stringency with a 1.65-kb HinfI fragment of the 4.2-kb plasmid.     

Mechanism of integration of the broad-host-range plasmid RP4 into the chromosome of Myxococcus xanthus

The site-specific recombination mechanism through which the plasmid RP4 has been previously shown to integrate into the chromosome of Myxococcus xanthus has been investigated further. Once integrated in one of the numerous chromosomal sites from two different strains, through a precise site on the plasmid, the latter can be excised either precisely or after a definite 14.5-kb deletion. In some cases, the integration is followed by different DNA rearrangements that yield a higher rate of excision and integration. A model for the site-specific integration and excision of the plasmid is proposed.     

A Family of Telomere-Associated Autonomously Replicating Sequences and Their Functions in Targeted Recombination in Hansenula polymorpha DL-1

A family of multiple autonomously replicating sequences (ARSs) which are located at several chromosomal ends of Hansenula polymorpha DL-1 has been identified and characterized. Genomic Southern blotting with an ARS, HARS36, originating from the end of a chromosome, as a probe showed several homologues in the genome of H. polymorpha. Nucleotide sequences of the three fragments obtained by a selective cloning for chromosomal ends were nearly identical to that of HARS36. All three fragments harbored an ARS motif and ended with 18 to 23 identical repetitions of 5′-GGGTGGCG-3′ which resemble the telomeric repeat sequence in other eukaryotes. Transformation of H. polymorpha with nonlinearized plasmids containing the newly obtained telomeric ARSs almost exclusively resulted in the targeted integration of a single copy or multiple tandem copies of the plasmid into the chromosomes. The sensitivity to exonuclease Bal31 digestion of the common DNA fragment in all integrants confirmed the telomeric origin of HARS36 homologues, suggesting that several chromosomal ends, if not all of them, consisted of the same ARS motif and highly conserved sequences observed in HARS36. Even though the frequencies of targeted recombination were varied among the ends of the chromosomes, the overall frequency was over 96%. The results suggested that the integration of the plasmids containing telemeric ARSs occurred largely through homologous recombination at the telomeric repeats, which serve as high-frequency recombination targets.     

A plasmid vector allowing positive selection of recombinant plasmids in Streptococcus pneumoniae

A new plasmid, pSP2, was constructed as a cloning vector for use in Streptococcus pneumoniae. It allows direct selection of recombinant plasmids, even for DNA fragments not homologous to the S. pneumoniae chromosome, as based on the failure to maintain long inverted repeats (LIRs) hyphen-free in bacterial plasmids. Plasmid pSP2 contains a 1.4-kb BamHI fragment ("hyphen") flanked by 1.9-kb LIRs. The removal of the 1.4-kb BamHI fragment followed by ligation creates a plasmid containing a 1.9-kb insert-free LIR; plasmids with such non-hyphenated LIRs were not established when transferred into S. pneumoniae. Replacement of the original 1.4-kb insert by other restriction fragments restored plasmid viability. Investigation of plasmid transfer by transformation suggests that intrastrand synapsis between the LIRs could occur, thus facilitating plasmid establishment (a process we call self-facilitation). Such an intrastrand synapsis could also account for rare occurrences of insert-inversion noticed upon transfer as well as for the formation of palindrome-deleted derivatives at low frequency. Plasmid pSP2 carries two selectable genes, tet and ermC, and can be used for cloning of fragments produced by a variety of restriction enzymes (BamHI, Bg/II, Bc/I or Sau3A, and Sa/I or XhoI).     

Mechanism of integration of the broad-host-range plasmid RP4 into the chromosome of Myxococcus xanthus

The site-specific recombination mechanism through which the plasmid RP4 has been previously shown to integrate into the chromosome of Myxococcus xanthus has been investigated further. Once integrated in one of the numerous chromosomal sites from two different strains, through a precise site on the plasmid, the latter can be excised either precisely or after a definite 14.5-kb deletion. In some cases, the integration is followed by different DNA rearrangements that yield a higher rate of excision and integration. A model for the site-specific integration and excision of the plasmid is proposed.     

Illegitimate recombination in Bacillus subtilis: nucleotide sequences at recombinant DNA junctions

Summary The illegitimate integration of plasmid pGG20 (the hybrid between Staphylococcus aureus plasmid pE194 and Escherichia coli plasmid pBR322) into the Bacillus subtilis chromosome was studied. It was found that nucleotide sequences of both parental plasmids could be involved in this process. The recombinant DNA junctions between plasmid pGG20 and the chromosome were cloned and their nucleotide sequences were determined. The site of recombination located on the pBR322 moiety carried a short region (8 bp) homologous with the site on the chromosome. The nucleotide sequences of the pE194 recombination sites did not share homology with chromosomal sequences involved in the integration process. Two different pathways of illegitimate recombination in B. subtilis are suggested.