Bacterial natural transformation by highly fragmented and damaged DNA.

DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often <100 bp) and may persist in the environment for more than half a million years. Fragmented DNA is recognized as nutrient source for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥ 20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations generated by uptake of short DNA fragments escape mismatch repair. Moreover, double-nucleotide polymorphisms appear more common among genomes of transformable than nontransformable bacteria. Our findings reveal that short and damaged, including truly ancient, DNA molecules, which are present in large quantities in the environment, can be acquired by bacteria through natural transformation. Our findings open for the possibility that natural genetic exchange can occur with DNA up to several hundreds of thousands years old.

Molecular aspects of gene transfer and foreign DNA acquisition in prokaryotes with regard to safety issues.

Horizontal gene transfer (HGT) is part of prokaryotic life style and a major factor in evolution. In principle, any combinations of genetic information can be explored via HGT for effects on prokaryotic fitness. HGT mechanisms including transformation, conjugation, transduction, and variations of these plus the role of mobile genetic elements are summarized with emphasis on their potential to translocate foreign DNA. Complementarily, we discuss how foreign DNA can be integrated in recipient cells through homologous recombination (HR), illegitimate recombination (IR), and combinations of both, site-specific recombination, and the reconstitution of plasmids. Integration of foreign DNA by IR is very low, and combinations of IR with HR provide intermediate levels compared to the high frequency of homologous integration. A survey of studies on potential HGT from various transgenic plants indicates very rare transfer of foreign DNA. At the same time, in prokaryotic habitats, genes introduced into transgenic plants are abundant, and natural HGT frequencies are relatively high providing a greater chance for direct transfer instead of via transgenic plants. It is concluded that potential HGT from transgenic plants to prokaryotes is not expected to influence prokaryotic evolution and to have negative effects on human or animal health and the environment.

Double illegitimate recombination events integrate DNA segments through two different mechanisms during natural transformation of Acinetobacter baylyi.

Acquisition of foreign DNA by horizontal gene transfer is seen as a major source of genetic diversity in prokaryotes. However, strongly divergent DNA is not genomically integrated by homologous recombination and would depend on illegitimate recombination (IR) events which are rare. We show that, by two mechanisms, during natural transformation of Acinetobacter baylyi two IR events can integrate DNA segments. One mechanism is double illegitimate recombination (DIR) acting in the absence of any homology (frequency: 7 x 10(-13) per cell). It occurs about 10(10)-fold less frequent than homologous transformation. The other mechanism is homology-facilitated double illegitimate recombination (HFDIR) being about 440-fold more frequent (3 x 10(-10) per cell) than DIR. HFDIR depends on a homologous sequence located between the IR sites and on recA(+). In HFDIR two IR events act on the same donor DNA molecule as shown by the joint inheritance of molecular DNA tags. While the IR events in HFDIR occurred at microhomologies, in DIR microhomologies were not used. The HFDIR phenomenon indicates that a temporal recA-dependent association of donor DNA at a homology in recipient DNA may facilitate two IR events on the 5' and 3' heterologous parts of the transforming DNA molecule.

Effects of single-strand DNases ExoI, RecJ, ExoVII, and SbcCD on homologous recombination of recBCD+ strains of Escherichia coli and roles of SbcB15 and XonA2 ExoI mutant enzymes.

To assess the contributions of single-strand DNases (ssDNases) to recombination in a recBCD+ background, we studied 31 strains with all combinations of null alleles of exonuclease I (delta xon), exonuclease VII (xseA), RecJ DNase (recJ), and SbcCD DNase (sbcCD) and exonuclease I mutant alleles xonA2 and sbcB15. The xse recJ sbcCD delta xon and xse recJ sbcCD sbcB15 quadruple mutants were cold sensitive, while the quadruple mutant with xonA2 was not. UV sensitivity increased with ssDNase deficiencies. Most triple and quadruple mutants were highly sensitive. The absence of ssDNases hardly affected P1 transductional recombinant formation, and conjugational recombinant production was decreased (as much as 94%) in several cases. Strains with sbcB15 were generally like the wild type. We determined that the sbcB15 mutation caused an A183V exchange in exonuclease motif III and identified xonA2 as a stop codon eliminating the terminal 8 amino acids. Purified enzymes had 1.6% (SbcB15) and 0.9% (XonA2) of the specific activity of wild-type Xon (Xon+), respectively, with altered activity profiles. In gel shift assays, SbcB15 associated relatively stably with 3' DNA overhangs, giving protection against Xon+. In addition to their postsynaptic roles in the RecBCD pathway, exonuclease I and RecJ are proposed to have presynaptic roles of DNA end blunting. Blunting may be specifically required during conjugation to make DNAs with overhangs RecBCD targets for initiation of recombination. Evidence is provided that SbcB15 protein, known to activate the RecF pathway in recBC strains, contributes independently of RecF to recombination in recBCD+ cells. DNA end binding by SbcB15 can also explain other specific phenotypes of strains with sbcB15.

A double kill gene cassette for the positive selection of transforming non-selective DNA segments in Acinetobacter baylyi BD413.

Natural transformation has been widely used for the monitoring of DNA in biological and environmental samples. These assays depended on selectable traits on the tested DNA. We have now developed a transformation assay system in which recombinational removal of a cassette with two conditional kill genes (hok and sacB) from the recipient genome provides positive selection for non-selective DNA. The cassette was integrated into the Acinetobacter baylyi BD413 chromosome within trpE and was flanked by two segments of non-selective test DNA, which in this study were from a T-DNA construct previously used to generate a transgenic potato. Genes for tetracycline and spectinomycin/streptomycin resistance located at the sides of the cassette allowed to maintain selection pressure against spontaneous loss of the cassette. Plasmid DNA containing the T-DNA gave transformation frequencies ranging linearly from 10(-4) per recipient (at 1 mug DNA ml(-1)) down to 10(-7) (1 ng DNA ml(-1)) by selecting for survivors after activation of both kill functions. Transformation depended on the two flanking homologous segments for recombinational exchange. DNA of the transgenic potato also gave positive scores in spite of the about 10(5)-fold dilution of T-DNA by potato DNA. False positives having a spontaneous deletion of hok and sacB occurred at a frequency of 1.8x10(-9) per cell but could be distinguished by PCR from real transformants. Thus, the system is suitable for detection of transformation frequencies down to about 10(-9). Hok and sacB as well as the regulatory system used (LacI-lac operator and T5 promoter) are known to function in many organisms suggesting wide applicability of the cassette for positive selection.

Natural transformation of Pseudomonas stutzeri by single-stranded DNA requires type IV pili, competence state and comA.

Pseudomonas stutzeri, in addition to being transformed by duplex DNA, is also transformed by the sense or antisense strand of the genetic marker employed (hisX(+)) or by heat-denatured chromosomal DNA. Transformation was absent in non-competent cells and in mutants defective for pilus biogenesis (pilA, pilC) and function (pilT) or DNA translocation into the cytoplasm (comA). Uptake of (3)H-thymidine-labeled single-stranded DNA was hardly detectable reflecting the 20- to 60-fold lower transformation compared to duplex DNA. The results suggest that the steps in natural transformation also accommodate single-stranded DNA and that DNA translocation from the periplasm into the cytoplasm is not necessarily coupled to the degradation of a complementary strand. Small DNA single-stranded fragments are thus not excluded from horizontal gene transfer by transformation.

The RecBCD and SbcCD DNases suppress homology-facilitated illegitimate recombination during natural transformation of Acinetobacter baylyi.

During natural transformation of Acinetobacter baylyi, the genomic integration of foreign (non-homologous) DNA is possible when the DNA contains a single segment homologous to the recipient genome (anchor) through homologous recombination in the anchor facilitating illegitimate recombination in the neighbouring foreign DNA (homology-facilitated illegitimate recombination; HFIR). DNA integration by HFIR occurs about 10 000 times less frequently than fully homologous recombination, but at least 100 000-fold more frequently than integration in the absence of any homology. We investigated the influence of the RecBCD enzyme (DNase/helicase) and SbcCD DNase (DNA-structure-specific single-strand endonuclease and exonuclease) on HFIR. In a recBCD null mutant the acquisition of foreign DNA was elevated 11-fold relative to wild-type cells by a 6.9-fold increased HFIR frequency and by the integration of longer stretches of foreign DNA in each event. In an sbcCD null mutant, the foreign DNA acquisition was 4.5-fold higher than in the wild-type, while homologous transformation with large DNA molecules was unaffected and increased 3.2-fold with small DNA fragments. The sbcCD mutation partially suppressed the high UV sensitivity and low viability of the recBCD mutant and also decreased its foreign DNA acquisition by HFIR to the lower level of the sbcCD mutant. We propose that suppression of HFIR results from the elimination of double-stranded intermediates of the HFIR process during transformation by RecBCD, and by SbcCD interfering with branched molecules. Our results provide evidence that the homologous recombination enzymes RecBCD and SbcCD control the level of foreign DNA acquisition by HFIR.

Frequent integration of short homologous DNA tracks during Acinetobacter baylyi transformation and influence of transcription and RecJ and SbcCD DNases.

The minimal length of integrated homologous donor DNA tracks in Acinetobacter baylyi transformation and factors influencing the location and length of tracks were determined. Donor DNA contained the nptII gene region (kanamycin resistance, KmR). This region carried nine approximately evenly spaced silent nucleotide sequence tags and was embedded in heterologous DNA. Recipient cells carried the normal nptII gene with a central 10 bp deletion (kanamycin-sensitive). The Km(R) transformants obtained had donor DNA tracks integrated covering on average only 4.6 (2-7) of the nine tags, corresponding to about 60 % of the 959 nt homologous donor DNA segment. The track positions were biased towards the 3' end of nptII. While the replication direction of recipient DNA did not affect track positions, inhibited transcription (by rifampicin) shifted the beginning of tracks towards the nptII promoter. Absence of the RecJ DNase decreased the length of tracks. Absence of SbcCD DNase increased the integration frequency of the 5' part of nptII, which can form hairpin structures of 43-75 nt, suggesting that SbcCD DNase interferes with hairpins in transforming DNA. In homology-facilitated illegitimate recombination events during transformation (in which a homologous DNA segment serves as a recombinational anchor to facilitate illegitimate recombination in neighbouring heterologous DNA), on average only about half of the approximately 800 nt long tagged nptII anchor sequences were integrated. From donor DNA with an approximately 5000 nt long homologous segment having the nptII gene in the middle, most transformants (74 %) had only a part of the donor nptII integrated, showing that short track integration occurs frequently also from large homologous DNA. It is discussed how short track integration steps can also accomplish incorporation of large DNA molecules.

Deletions of recBCD or recD influence genetic transformation differently and are lethal together with a recJ deletion in Acinetobacter baylyi.

In prokaryotes, homologous recombination is essential for the repair of genomic DNA damage and for the integration of DNA taken up during horizontal gene transfer. In Escherichia coli, the exonucleases RecJ (specific for 5' single-stranded DNA) and RecBCD (degrades duplex DNA) play important roles in recombination and recombinational double-strand break (DSB) repair by the RecF and RecBCD pathways, respectively. The cloned recJ of Acinetobacter baylyi partially complemented an E. coli recJ mutant, suggesting functional similarity of the enzymes. A DeltarecJ mutant of A. baylyi was only slightly altered in transformability and was not affected in UV survival. In contrast, a DeltarecBCD mutant was UV-sensitive, and had a low viability and altered transformation. Compared to wild-type, transformation with large chromosomal DNA fragments was decreased about 5-fold, while transformation with 1.5 kbp DNA fragments was increased 3.3- to 7-fold. A DeltarecD mutation did not affect transformation, viability or UV resistance. However, double mutants recJ recBCD and recJ recD were non-viable, suggesting that the RecJ DNase or the RecBCD DNase (presumably absent in recD) becomes essential for the recombinational repair of spontaneously inactivated replication forks if the other DNase is absent. A model of recombination during genetic transformation is discussed in which the two ends of the single-stranded donor DNA present in the cytoplasm frequently integrate separately and often with a time difference. If replication runs through that genomic region before both ends of the donor DNA are ligated to recipient DNA, a double-strand break (DSB) is formed. In these cases, transformation becomes dependent on DSB repair.

The RecJ DNase strongly suppresses genomic integration of short but not long foreign DNA fragments by homology-facilitated illegitimate recombination during transformation of Acinetobacter baylyi.

Homology-facilitated illegitimate recombination (HFIR) promotes genomic integration of foreign DNA with a single segment homologous to the recipient genome by homologous recombination in the segment accompanied by illegitimate fusion of the heterologous sequence. During natural transformation of Acinetobacter baylyi HFIR occurs at about 0.01% of the frequency of fully homologous recombination. The role of the 5' single-strand-specific exonuclease RecJ in HFIR was investigated. Deletion of recJ increased HFIR frequency about 20-fold compared with wild type while homologous recombination was not affected. Illegitimate fusion sites were predominantly located within 360 nucleotides away from the homology whereas in wild type most fusion sites were distal (500-2500 nucleotides away). RecJ overproduction reduced the HFIR frequency to half compared with wild type, and transformants with short foreign DNA segments were diminished, leading to on average 866 foreign nucleotides integrated per event (682 in wild type, 115 in recJ). In recJ always the 3' ends of donor DNA were integrated at the homology whereas in wild type these were 3' or 5'. RecJ apparently suppresses HFIR by degrading 5' non-homologous DNA tails at the post-synaptic stage. We propose that the RecJ activity level controls the HFIR frequency during transformation and the amount of foreign DNA integrated per event.

Impact of mutS inactivation on foreign DNA acquisition by natural transformation in Pseudomonas stutzeri.

In prokaryotic mismatch repair the MutS protein and its homologs recognize the mismatches. The mutS gene of naturally transformable Pseudomonas stutzeri ATCC 17587 (genomovar 2) was identified and characterized. The deduced amino acid sequence (859 amino acids; 95.6 kDa) displayed protein domains I to IV and a mismatch-binding motif similar to those in MutS of Escherichia coli. A mutS::aac mutant showed 20- to 163-fold-greater spontaneous mutability. Transformation experiments with DNA fragments of rpoB containing single nucleotide changes (providing rifampin resistance) indicated that mismatches resulting from both transitions and transversions were eliminated with about 90% efficiency in mutS+. The mutS+ gene of strain ATCC 17587 did not complement an E. coli mutant but partially complemented a P. stutzeri JM300 mutant (genomovar 4). The declining heterogamic transformation by DNA with 0.1 to 14.6% sequence divergence was partially alleviated by mutS::aac, indicating that there was a 14 to 16% contribution of mismatch repair to sexual isolation. Expression of mutS+ from a multicopy plasmid eliminated autogamic transformation and greatly decreased heterogamic transformation, suggesting that there is strong limitation of MutS in the wild type for marker rejection. Remarkably, mutS::aac altered foreign DNA acquisition by homology-facilitated illegitimate recombination (HFIR) during transformation, as follows: (i) the mean length of acquired DNA was increased in transformants having a net gain of DNA, (ii) the HFIR events became clustered (hot spots) and less dependent on microhomologies, which may have been due to topoisomerase action, and (iii) a novel type of transformants (14%) had integrated foreign DNA with no loss of resident DNA. We concluded that in P. stutzeri upregulation of MutS could enforce sexual isolation and downregulation could increase foreign DNA acquisition and that MutS affects mechanisms of HFIR.

Genomovars 11 to 18 of Pseudomonas stutzeri, identified among isolates from soil and marine sediment.

Amongst 440 strains of Pseudomonas stutzeri isolated from soil and marine sediment for a population genetic study, eight strains were each presumed to represent a novel genomic group and were compared with each other and to reference strains of P. stutzeri genomovars 1 to 10 and other Pseudomonas species by DNA-DNA hybridization, 16S rRNA and internally transcribed 16S-23S rRNA spacer region (ITS1) sequences and basic physiological properties defining the species. While 16S rRNA and ITS1 gene sequences positioned the eight strains within the phylogenetic branch of P. stutzeri, the DNA-DNA hybridizations with reference strains of the 10 described genomovars and among the novel strains were generally below 70 %, which is the threshold for species and genomovar differentiation. Since the physiological properties studied in the eight strains fitted the profile of P. stutzeri, eight new genomovars of P. stutzeri, numbered 11 to 18, are proposed, with strains 28a50, 28a39, 28a22, 28a3, 4C29, 24a13, 24a75 and MT-1 being the reference strains. The highly transformable reference strain 28a3 of genomovar 14 had a localized 16S rRNA gene sequence tag characteristic of genomovar strains 2 and 3, suggesting a possible horizontal gene transfer event involving part of the 16S rRNA gene.

Monitoring the spread of recombinant DNA from field plots with transgenic sugar beet plants by PCR and natural transformation of Pseudomonas stutzeri.

Previous studies had shown that recombinant DNA can be detected for several months in soil after the deposition of litter from transgenic (tg) plants. Here we show by PCR monitoring of field releases of tg sugar beet plants that during the growth of the plants the soil close to the plants and also plant material contains recombinant DNA, in the form of extracellular molecules. Surprisingly, the monitoring also revealed the presence of tg DNA in many field plots (30-70%) in which tg plants were never grown. These studies and the further monitoring during other tg sugar beet release experiments by PCR and a novel bioassay (measuring the transforming potential of recombinant DNA for Pseudomonas stutzeri) indicated that recombinant DNA was only detectable in the surface soil of field plots and their vicinity where flowering of the tg beet plants was allowed. Recombinant DNA was found in soil at a distance of 50 m from pollen-producing plants surrounded by a strip with hemp plants as a containment regime. It is concluded that recombinant DNA is deposited in soil during the growth of tg sugar beets and that a major mechanism of recombinant DNA spread in the environment is the dispersal of pollen which allows recombinant DNA to persist in the field plot for at least a year.

Mechanisms of homology-facilitated illegitimate recombination for foreign DNA acquisition in transformable Pseudomonas stutzeri.

Intra- and interspecific natural transformation has been observed in many prokaryotic species and is considered a fundamental mechanism for the generation of genetic variation. Recently, it has been described in detail how, in transformable Acinetobacter BD413 and Streptococcus pneumoniae, long stretches of nucleotides lacking homology were integrated into recipient genomes when they were linked on one side to a small piece of DNA with homology to resident DNA serving as a recA-dependent recombination anchor. Now, such homology-facilitated illegitimate recombination (HFIR) has also been detected in transformable Pseudomonas stutzeri. However, analysis of the recombinants revealed qualitative and quantitative differences in their generation compared with that in Acinetobacter BD413. In P. stutzeri, foreign DNA with an anchor sequence was integrated 105- to 106-fold less frequently than fully homologous DNA, but still at least 200-fold more frequently than without the anchor. The anchor sequence could be as small as 311 bp. Remarkably, in 98% of the events, the 3' end was integrated within the homologous anchor, whereas the 5' end underwent illegitimate fusion. Moreover, about one-third of the illegitimate fusion sites shared no or only a single identical basepair in foreign and resident DNA. The other fusions occurred within microhomologies of up to 6 bp with a higher GC content on average than the interacting nucleotide sequences. Foreign DNA of 69-1903 bp was integrated, and resident DNA of 22-2345 bp was lost. In a recA mutant, HFIR was not detectable. The findings suggest that genomic acquisition of foreign DNA by HFIR during transformation occurs widely in prokaryotes, but that details of the required recombination and strand fusion mechanisms may differ between organisms from different genera.

Identification of complex composition, strong strain diversity and directional selection in local Pseudomonas stutzeri populations from marine sediment and soils.

Members of Pseudomonas stutzeri have been isolated world-wide from various habitats including aquatic and terrestrial ecosystems. The global population has a clonal structure, is of exceptionally high genetic diversity and has been grouped into eight genomovars. We have analysed four local populations (n = 89-125) from three geographically separated habitats (two from a marine sediment and two from different soils) by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), restriction fragment length polymorphism (RFLP) of the rpoB gene and 16S rDNA sequences in order to quantify the influence of evolutionary forces on closely related groups of proliferating cells in situ. All populations consisted of a complex structure of genomic subgroups with variable numbers of members. The analyses revealed that the two populations from marine sediment were rather similar. At least three of the populations were influenced by migrational input as concluded from the presence of members from different genomovars. All populations showed very high strain diversity suggesting strong influence of mutability. Neutrality tests indicated that two or possibly three of the populations were shaped by directional selection. Thus, the local populations of P. stutzeri reflect already the high genetic diversity of the global population and are influenced, to different extents, by migration, mutation and directional selection.

Highly different levels of natural transformation are associated with genomic subgroups within a local population of Pseudomonas stutzeri from soil.

A highly sensitive and specific PCR-based method of monitoring 16S rRNA genes of Pseudomonas stutzeri was developed for searching P. stutzeri DNA in environmental samples. This monitoring was combined with a reliable and sensitive method for isolating P. stutzeri colony formers from soil and sediment, depending on their utilization of ethylene glycol, starch, and maltose. With these techniques, P. stutzeri populations (n = 2 to 170) were obtained from five of six sites giving positive PCR signals (including three marine sediment and two soil samples). The phylogenetic positions of isolates from the five sites, based on their 16S ribosomal DNA sequences, indicated that the environmental isolates were affiliated with different genomovars of P. stutzeri. Using the broad-host-range plasmid pNS1 with kanamycin and gentamicin resistance determinants as the transforming DNA, naturally transformable strains were identified among the isolates from all sites. For one population from soil, the genetic relationship of the 120 members was determined by randomly amplified polymorphic DNA-PCR with three PCR primers. Among the population members which are taxonomically closely related as determined by 16S sequence comparisons of group representatives, a rather high genetic diversity and a characteristic clustering into subgroups were found. Remarkably, within the population, nontransformability and different levels of transformability (a frequency between about 10(-9) and 10(-4) per cell) were often associated with distinct genetic subgroups. It is concluded that transformability is widespread among environmental P. stutzeri strains and that its specific level is a heritable trait that may vary strongly within a local population.

Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination.

The active uptake of extracellular DNA and its genomic integration is termed natural transformation and constitutes a major horizontal gene-transfer mechanism in prokaryotes. Chromosomal DNA transferred within a species can be integrated effectively by homologous recombination, whereas foreign DNA with low or no sequence homology would rely on illegitimate recombination events, which are rare. By using the nptII(+) gene (kanamycin resistance) as selectable marker, we found that the integration of foreign DNA into the genome of the Gram-negative Acinetobacter sp. BD413 during transformation indeed was at least 10(9)-fold lower than that of homologous DNA. However, integration of foreign DNA increased at least 10(5)-fold when it was linked on one side to a piece of DNA homologous to the recipient genome. Analysis of foreign DNA integration sites revealed short stretches of sequence identity (3-8 bp) between donor and recipient DNA, indicating illegitimate recombination events. These findings suggest that homologous DNA served as a recombinational anchor facilitating illegitimate recombination acting on the same molecule. Homologous stretches down to 183 nucleotides served as anchors. Transformation with heteroduplex DNA having different nucleotide sequence tags in the strands indicated that strands entered the cytoplasm 3' to 5' and that strands with either polarity were integrated by homologous recombination. The process led to the genomic integration of thousands of foreign nucleotides and often was accompanied by deletion of a roughly corresponding length of recipient DNA. Homology-facilitated illegitimate recombination would explain the introgression of DNA in prokaryotic genomes without the help of mobile genetic elements.

DNA restriction is a barrier to natural transformation in Pseudomonas stutzeri JM300.

Natural transformation is a mechanism for intra- and interspecific transfer of chromosomal DNA in Pseudomonas stutzeri. During this process a single strand derived from duplex DNA is transported into the cytoplasm and recombined with resident DNA. By electroporation, which introduces duplex DNA into cells, 100-fold lower transformation frequencies of P. stutzeri JM300 were observed with shuttle vector or broad-host-range plasmid DNA when the plasmids had replicated in Escherichia coli and not in P. stutzeri JM300. Moreover, the natural transformation with cloned chromosomal P. stutzeri JM300 DNA was reduced about 40-fold when the DNA had not been propagated in P. stutzeri JM300 but in E. coli. Restriction was also active during natural transformation by single-stranded DNA. Restriction during natural transformation and electroporation was abolished in mutants isolated from mutagenized JM300 cells after applying a multiple plasmid electroporation strategy for the enrichment of restriction-defective strains. The mutants had retained the ability for DNA modification. The P. stutzeri strain ATCC 17587 was found to have no restriction-modification system as seen in JM300. It is discussed whether restriction during natural transformation acts at presynaptic or postsynaptic stages of transforming DNA. Restriction as a barrier to transformation apparently contributes to sexual isolation and therefore may promote speciation in the highly diverse species P. stutzeri.

Natural genetic transformation of Pseudomonas stutzeri in a non-sterile soil.

Natural transformation of the soil bacterium Pseudomonas stutzeri JM300 in a non-sterile brown earth microcosm was studied. For this purpose, the microcosm was loaded with purified DNA (plasmid or chromosomal DNA, both containing a high-frequency-transformation marker, his+, of the P. stutzeri genome), the non-adsorbed DNA was washed out with soil extract and then the soil was charged with competent cells (his-1). Both chromosomal and plasmid transformants were found among the P. stutzeri cells recovered from the soil. The number of plasmid transformants increased in a linear fashion with the amount of DNA added [10-600 ng (0.7 g soil)-1]. The observed efficiency of transformation, the time course of transformant formation and the complete inhibition of transformation by DNase I, when added to the soil, were similar to that seen in optimized transformations in nutrient broth. Addition of cells as late as 3 d after loading the soil with plasmid DNA still yielded 3% of the initial transforming activity. This suggests that nucleases indigenous to the soil destroyed the transforming DNA, but at a rate allowing considerable DNA persistence. Transformants were also obtained when intact P. stutzeri cells were introduced into the soil to serve as plasmid DNA donors. Apparently, DNA was released from the cells, adsorbed to the soil material and subsequently taken up by recipient cells. The results indicate that competent cells of P. stutzeri were able to find access to and take up DNA bound on soil particles in the presence of micro-organisms and DNases indigenous to the soil.