Transformation by extracellular DNA produced by Pseudomonas aeruginosa

Most Pseudomonas aeruginosa strains are capable of producing extracellular DNA. Very closely linked chromosomal markers (leu+ and trp+) were co-transferred to P. aeruginosa PAO1819 (leu9001, trp9008) by the extracellular DNA produced by P. aeruginosa strains IFO3445 and PAO1 at a frequency of 10(-7) to 10(-8). Treatment of the extracellular DNA with DNase, heating at 95 C or sonication completely destroyed its transforming ability. The R plasmid in the extracellular DNA produced by P. aeruginosa IFO3445 (RP4) or PAO2142 (RLb679) could be transferred to Escherichia coli ML4901 or P. aeruginosa PAO1819. The resultant transformants showed identical resistance patterns in the respective donors, and the sizes of the DNAs of RLb679 and RP4 isolated from the transformants were the same as those in the respective donors. These results demonstrate that the extracellular DNA contains both chromosomal DNA and plasmid DNA, and that it exhibits transforming ability. This implies that transformation by the extracellular DNA produced by P. aeruginosa may occur in nature and this seems to be of clinical importance in view of the spread of R plasmids among pathogens.     

Integration of the RP4 factor with the chromosome in Escherichia coli K12 and the formation of 2 types of Hfr-strains]

The mutant RP4ts12, derived from the R-factor RP4 and thermosensitive in replication, is incorporated into the chromosome A3dna(ts) of E. coli K12, thus suppressing dnaA mutation. The integration of this factor into the chromosome leads to the formation of Hfr strains of two types: the strains of the first type transfer plasmid markers to recipient cells earlier than to chromosomal ones; the strains of the second type transfer plasmid markers to recipient cells after chromosomal ones. During conjugation the R-factor integrated into the chromosome dissociates from chromosomal DNA introduced into the recipient cell and becomes autonomous.     

Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin ( oriA ). Transformants were recovered only with the plasmid containing oriA , and all transformants contained an integrated plasmid copy at oriA , suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain.

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Duplication insertion of drug resistance determinants in the radioresistant bacterium Deinococcus radiodurans.

Escherichia coli drug resistance plasmids were introduced into Deinococcus radiodurans by cloning D. radiodurans DNA into the plasmids prior to transformation. The plasmids were integrated into the chromosome of the transformants and flanked by a direct repeat of the cloned D. radiodurans segment. The plasmid and one copy of the flanking chromosomal segment constituted a unit ("amplification unit") which was found repeated in tandem at the site of chromosomal integration. Up to 50 copies of the amplification unit were present per chromosome, accounting for approximately 10% of the genomic DNA. Circular forms of the amplification unit were also present in D. radiodurans transformants. These circles were introduced into E. coli, where they replicated as plasmids. The drug resistance determinants which have been introduced into D. radiodurans in this fashion are cat (from Tn9) and aphA (from Tn903). Transformation of D. radiodurans to drug resistance was efficient when the donor DNA was from D. radiodurans or E. coli, but was greatly reduced when the donor DNA was linearized with restriction enzymes prior to transformation. In the course of the study, a plasmid, pS16, was discovered in D. radiodurans R1, establishing that all Deinococcus strains so far examined contain plasmids.     

Transposon Mutagenesis and Excision of R' Plasmids by Conjugative, Chimeric Plasmid pUW942 in Extra-Slow-Growing Rhizobium japonicum Strains

Transposons Tn501 (specifying mercury resistance) and Tn7 (specifying resistance to trimethoprim and streptomycin) were introduced into extra-slow-growing Rhizobium japonicum by conjugal transfer of the 82 kilobase chimeric plasmid pUW942. Mercury-resistant transconjugants were obtained at a frequency of 10 to 10. The transfer frequency of streptomycin resistance was lower than that of mercury resistance, and Tn7 was relatively unstable. pUW942 was not maintained as an autonomously replicating plasmid in R. japonicum strains. However, some of the Hg transconjugants from the RJ19FY, RJ17W, and RJ12S strains acquired antibiotic markers of the vector plasmid pUW942. Southern hybridization of plasmid and chromosomal DNA of R. japonicum strains with P-labeled pUW942 and pAS8Rep-1, the same plasmid as pUW942 except that it does not contain Tn501, revealed the formation of cointegrates between pUW942 and the chromosome of R. japonicum. More transconjugants with only Tn501 insertions in plasmids or the chromosome were obtained in crosses with strains RJ19FY and RJ17W than with RJ12S. These retained stable Hg both in plant nodules and under nonselective in vitro growth conditions. One of the RJ19FY and two of the RJ12S Hg transconjugants with vector plasmid-chromosome cointegrates conjugally transferred plasmids of 82, 84 or 86, and 90 kilobases, respectively, into plasmidless Escherichia coli C. These plasmids strongly hybridized to pUW942 and EcoRI digests of total DNA of each respective R. japonicum strain but not to indigenous plasmid DNA of the R. japonicum strains. These R' plasmids consisted of pUW942-specific EcoRI fragments and an additional one or two new fragments derived from the R. japonicum chromosome.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum.

Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.     

Electrotransformation ofStreptococcus sanguis Challis

Plasmid DNA was introduced into noncompetent cells ofStreptococcus sanguis Challis by an electrotransformation technique. The procedure was simple and rapid, did not require elaborate pretreatment of cells, and yielded transformant colonies in 24 h. The maximum transformation efficiency attained was 2.1×10⁴ transformants per g of pVA736. Molecular rearrangements and deletions were not detected in plasmid DNA isolated from transformants.     

Transfer of plasmid RP4 to Myxococcus xanthus and evidence for its integration into the chromosome.

The broad-host-range plasmid RP4 and its derivative R68.45 were transferred to Myxococcus xanthus DK101 and DZ1; RP4 was maintained integrated in the chromosome. Loss of plasmid markers occurred during the growth of the transconjugants, which could be prevented by selective pressure with oxytetracycline. The integrated plasmid was transferred back to Escherichia coli often as RP4-prime plasmids carrying various segments of the M. xanthus chromosome. It also mediated chromosomal transfer between M. xanthus strains.     

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.     

Direct and indirect gene replacements in Aspergillus nidulans.

We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC- strain to trpC+. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC- allele in a minority of the strains. An A. nidulans trpC+ gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC- argB+ strain to trpC+. Approximately 30% of the transformants were simultaneously argB-. The null argB allele had replaced the wild-type allele in a majority of these strains. The A. nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC- strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC- progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae.     

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.     

Site-specific integration in Saccharopolyspora erythraea and multisite integration in Streptomyces lividans of actinomycete plasmid pSE101.

An 11.3-kilobase-pair plasmid, designated pSE101, exists in Saccharopolyspora erythraea NRRL 2338 as an integrated sequence (pSE101int) at a unique chromosomal location and in the free form in less than an average of 1 copy per 10 chromosomes. The plasmid sequence is missing from S. erythraea NRRL 2359. Restriction maps of the free and integrated forms of pSE101 showed point-to-point correspondence. Plasmid pECT2 was constructed by ligation of pSE101, pBR322, and the gene for thiostrepton resistance (tsr). When introduced by polyethylene glycol-mediated transformation into protoplasts of S. erythraea NRRL 2359, all thiostrepton-resistant regenerants examined were found to carry a single copy of pECT2 in the integrated state at a single chromosomal site. The chromosomal site of pECT2 integration in strain NRRL 2359 (attB) corresponded to the chromosomal location of pSE101int in strain NRRL 2338. The plasmid crossover site (attP) was mapped to the plasmid site that corresponded to the site of interruption of the plasmid sequence in the host carrying pSE101int, indicating that site-specific integrative recombination had occurred. An additional 2.8-kilobase-pair chromosomal sequence homologous to a segment of pSE101 was also observed in strains NRRL 2338 and NRRL 2359. After introduction of pECT2 into Streptomyces lividans, approximately half of the transformants examined were found to carry the plasmid as a stable, autonomously replicating element. The other half carried a single copy of pECT2 as an integrated sequence, but the location of pECT2int in Streptomyces lividans varied from one transformant to another. In each case, integrative crossover used the attP site. A model is proposed to account for the determination of the particular state of pSE101 in Streptomyces lividans.     

Transformation of Actinomyces spp. by a gram-negative broad-host-range plasmid.

The gram-negative broad-host-range vector pJRD215 was transferred by electroporation into strains of Actinomyces viscosus or Actinomyces naeslundii at efficiencies which ranged from 10(2) to 10(7) transformants per microgram of plasmid DNA. The Actinomyces transformants expressed pJRD215-encoded resistance to kanamycin and streptomycin. Moreover, the transforming plasmid DNA had not undergone any deletions or rearrangements, nor had it integrated into the genomes of these strains.     

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.     

Chromosome transfer and R-prime formation by an RP4::mini-Mu derivative in Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Proteus mirabilis

We have introduced into the wide host range conjugative plasmid RP4, a mini-Mu derivative which was known to be able to transpose spontaneously in E. coli K-12, and to induce in such a host several kinds of chromosomal rearrangements including replicon fusions. Unlike RP4, RP4::mini-Mu can mediate the transfer of the host chromosome to a recipient bacterium and generate R primes at high frequencies (10^?4 for the transfer of a given marker, 10^?5 for the formation of R primes carrying a given marker). Two such RP4::mini-Mu plasmids were introduced into one Salmonella typhimurium strain, one Klebsiella pneumoniae strain, and one Proteus mirabilis strain. Each of these three strains were mated with an E. coli K-12 recipient and transconjugants carrying R primes were recovered in all three cases at frequencies ranging from 5 × 10^?6 to 10^?7.     

Cloning of methylated transforming DNA from Neurospora crassa in Escherichia coli.

An arg-2 mutant of Neurospora crassa was transformed to prototrophy with a pBR322-N. crassa genomic DNA library. Repeated attempts to recover the integrated transforming DNA or segments thereof by digestion, ligation, and transformation of Escherichia coli, with selection for the plasmid marker ampicillin resistance, were unsuccessful. Analyses of a N. crassa transformant demonstrated that the introduced DNA was heavily methylated at cytosine residues. This methylation was shown to be responsible for our inability to recover transformants in standard strains of E. coli; transformants were readily obtained in a strain which is deficient in the two methylcytosine restriction systems. Restriction of methylated DNA in E. coli may explain the general failure to recover vector or transforming sequences from N. crassa transformants.     

Replication control of IncP plasmids

Clones of Escherichia coli with a chromosomally integrated RP4-prime plasmid were isolated and characterized. Chromosome transfer was increased about 50-fold and the Hfr still carried an autonomous plasmid indistinguishable from the original RP4-prime. This could be eliminated by pRP64 or R751, two distinguishably marked incompatible plasmids, giving rise to strains which stably retained the resistance patterns of both plasmids and which continued to transfer the chromosome at enhanced levels. In both cases, however, the copy number of the autonomous plasmid was reduced by the presence of a chromosomal RP4 such that the total number of P plasmid genomes (integrated and autonomous) remained constant. The results are consistent with the idea that copy number is controlled by diffusible inhibitors or initiators of replication.     

Transposition of the deo operon structural genes in Escherichia coli K-12 to plasmid RP4 using bacteriophage mu]

Transposition of the structural genes of the deo operon of Escherichia coli K-12 into plasmid RP4 by means of temperate bacteriophage Mu was carried out. Some variants of composite RP4-deo-Mu plasmids were obtained and the expression of the deo genes integrated into the RP4 plasmid genome was studied. It was shown that the expression of these genes remains under the control of the chromosomal regulatory genes (deoR and cytR); although the activity of thymidine phosphorilase in the strain E. coli which contains hybrid plasmid is 4-6 fold greater than that in strains of E. coli with chromosomal localization of the deo operon.