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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Transfer of plastid DNA from tobacco to the soil bacterium Acinetobacter sp. by natural transformation.

Acquisition of new genetic information by horizontal gene transfer is a major mechanism of genetic adaptation and evolution in prokaryotes. Naturally transformable cells of Acinetobacter sp. were exposed to plant DNA from leaf and root tissue of transplastomic tobacco.

Spread of recombinant DNA by roots and pollen of transgenic potato plants, identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp.

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.

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.

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.

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.

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.

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.

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.

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.

Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community.

In a field release experiment, rifampicin resistant mutants of two antagonistic plant-associated bacteria were used for seed tuber inoculation of transgenic T4 lysozyme expressing potatoes, transgenic control potatoes and non-transgenic parental potatoes. The T4 lysozyme tolerant Pseudomonas putida QC14-3-8 was originally isolated from the tuber surface (geocaulosphere) of T4 lysozyme producing plants and showed in vitro antibacterial activity to the bacterial pathogen Erwinia carotovora ssp. atroseptica.

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).