dOuter membrane vesicles (OMVs) are continually released from a range of bacterial species. Numerous functions of OMVs, including the facilitation of horizontal gene transfer (HGT) processes, have been proposed. In this study, we investigated whether OMVs contribute to the transfer of plasmids between bacterial cells and species using Gram-negative Acinetobacter baylyi as a model system. OMVs were extracted from bacterial cultures and tested for the ability to vector gene transfer into populations of Escherichia coli and A. baylyi, including naturally transformation-deficient mutants of A. baylyi. Anti-double-stranded DNA (anti-dsDNA) antibodies were used to determine the movement of DNA into OMVs. We also determined how stress affected the level of vesiculation and the amount of DNA in vesicles. OMVs were further characterized by measuring particle size distribution (PSD) and zeta potential. Transmission electron microscopy (TEM) and immunogold labeling were performed using antifluorescein isothiocyanate (anti-FITC)-conjugated antibodies and anti-dsDNA antibodies to track the movement of FITC-labeled and DNA-containing OMVs. Exposure to OMVs isolated from plasmid-containing donor cells resulted in HGT to A. baylyi and E. coli at transfer frequencies ranging from 10 ؊6 to 10 ؊8 , with transfer efficiencies of approximately 10 3 and 10 2 per g of vesicular DNA, respectively. Antibiotic stress was shown to affect the DNA content of OMVs as well as their hydrodynamic diameter and zeta potential. Morphological observations suggest that OMVs from A. baylyi interact with recipient cells in different ways, depending on the recipient species. Interestingly, the PSD measurements suggest that distinct size ranges of OMVs are released from A. baylyi.

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, doublenucleotide 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. microbial evolution | horizontal gene transfer | DNA degradation | early life | anachronistic evolution

We have investigated to what extent natural transformation acting on free DNA substrates can facilitate transfer of mobile elements including transposons, integrons and/or gene cassettes between bacterial species. Naturally transformable cells of Acinetobacter baylyi were exposed to DNA from integron-carrying strains of the genera Acinetobacter, Citrobacter, Enterobacter, Escherichia, Pseudomonas, and Salmonella to determine the nature and frequency of transfer. Exposure to the various DNA sources resulted in acquisition of antibiotic resistance traits as well as entire integrons and transposons, over a 24 h exposure period. DNA incorporation was not solely dependent on integrase functions or the genetic relatedness between species. DNA sequence analyses revealed that several mechanisms facilitated stable integration in the recipient genome depending on the nature of the donor DNA; homologous or heterologous recombination and various types of transposition (Tn21-like and IS26-like). Both donor strains and transformed isolates were extensively characterized by antimicrobial susceptibility testing, integron- and cassette-specific PCRs, DNA sequencing, pulsed field gel electrophoreses (PFGE), Southern blot hybridizations, and by re-transformation assays. Two transformant strains were also genome-sequenced. Our data demonstrate that natural transformation facilitates interspecies transfer of genetic elements, suggesting that the transient presence of DNA in the cytoplasm may be sufficient for genomic integration to occur. Our study provides a plausible explanation for why sequence-conserved transposons, IS elements and integrons can be found disseminated among bacterial species. Moreover, natural transformation of integron harboring populations of competent bacteria revealed that interspecies exchange of gene cassettes can be highly efficient, and independent on genetic relatedness between donor and recipient. In conclusion, natural transformation provides a much broader capacity for horizontal acquisitions of genetic elements and hence, resistance traits from divergent species than previously assumed.

Transgenic potato plants with the nptII gene coding for neomycin phosphotransferase (kanamycin resistance) as a selection marker were examined for the spread of recombinant DNA into the environment. We used the recombinant fusion of nptII with the tg4 terminator for a novel biomonitoring technique. This depended on natural transformation of Acinetobacter sp. strain BD413 cells having in their genomes a terminally truncated nptII gene (nptII; kanamycin sensitivity) followed by the tg4 terminator. Integration of the recombinant fusion DNA by homologous recombination in nptII and tg4 restored nptII, leading to kanamycin-resistant transformants. DNA of the transgenic potato was detectable with high sensitivity, while no transformants were obtained with the DNA of other transgenic plants harboring nptII in different genetic contexts. The recombinant DNA was frequently found in rhizosphere extracts of transgenic potato plants from field plots. In a series of field plot and greenhouse experiments we identified two sources of this DNA: spread by roots during plant growth and by pollen during flowering. Both sources also contributed to the spread of the transgene into the rhizospheres of nontransgenic plants in the vicinity. The longest persistence of transforming DNA in field soil was observed with soil from a potato field in 1997 sampled in the following year in April and then stored moist at 4°C in the dark for 4 years prior to extract preparation and transformation. In this study natural transformation is used as a reliable laboratory technique to detect recombinant DNA but is not used for monitoring horizontal gene transfer in the environment.Molecular techniques have been used now for about 2 decades to introduce new traits such as resistance to diseases, pests, and herbicides into plants of agronomical importance. Many of the transgenic plants contain antibiotic resistance genes, which were used as selection markers during their construction (9). The use of transgenic plants in agriculture leads to the presence of recombinant DNA in the environment on a large scale. The concern has been raised that an unintended transfer of the recombinant genetic material into the soil microbiota may occur and increase, e.g., antibiotic resistance in bacteria, including human pathogens (7,24,43).At the same time, the recombinant and thus specific nucleotide sequences of the DNA of genetically modified organisms enabled the quantitative tracing of the fate of DNA from transgenic organisms in the environment by applying PCR amplification. DNA of high molecular weight has been found to be present in soil sites where free DNA (10) or plant material (10, 29, 41) had been deposited and to persist in nonsterile soils for several months (33,34,41,42). It was suggested that DNA released from eukaryotic and prokaryotic cells constitutes an extracellular gene pool which can be used by naturally competent bacterial cells that take up DNA and integrate it into their genomes (natural transformation) (19,40). In microcosm experiments transforma...

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 59 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 DrecJ mutant of A. baylyi was only slightly altered in transformability and was not affected in UV survival. In contrast, a DrecBCD 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 DrecD 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.