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.T ransformation is considered a major horizontal genetransfer mechanism contributing to genetic adaptation and evolution of prokaryotes (1-3). Among the transformable organisms, the two species Neisseria gonorrhoeae and Haemophilus influenzae take up preferentially DNA of their own species recognized by specific nucleotide sequences (4, 5), whereas other species such as Streptococcus pneumoniae, Bacillus subtilis, and Acinetobacter sp. do not discriminate between their own and foreign DNA (2). With sufficient nucleotide-sequence similarity between donor DNA and the recipient genome, integration can occur by homologous recombination leading to a replacement of alleles or the integration of heterologous sequences when bracketed by homologous regions. However, the efficiency of homologous recombination decreases strongly with decreasing nucleotide-sequence similarity (6, 7).For the integration of foreign DNA with low or no sequence identity in bacteria, two basically different mechanisms have been identified. One relies on short specific nucleotide sequences recognized by cognate enzymes such as transposases or integrases that can cut and paste DNA at these sites. Nucleotide sequences bordered by such sites constitute genetic elements, examples being gene cassettes and integrons (8), transposons a...

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 developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in transgenic plant genomes. The assay is based on the recombinational repair of an nptII gene with an internal 10-bp deletion located on a plasmid downstream of a bacterial promoter. Uptake of an nptII gene by transformation restores kanamycin resistance. In Escherichia coli, promoterless nptII genes provided by electroporation were rescued with high efficiency in a RecA-dependent recombinational process. For the rescue of nptII genes present in chromosomal plant DNA, the system was adapted to natural transformation, which favours the uptake of linear DNA. When competent Acinetobacter sp. BD413 (formerly A. calcoaceticus) cells containing the mutant nptII gene on a plasmid were transformed with DNA from various transgenic plants carrying nptII as a marker gene (Solanum tuberosum, Nicotiana tabacum, Beta vulgaris, Brassica napus, Lycopersicon esculentum), kanamycin-resistant transformants were obtained roughly in proportion to the concentration of nptII genes in the plant DNA. The rescue of nptII genes occurred in the presence of a more than 6 x 10(6)-fold excess of plant DNA. Only 18 ng of potato DNA (2.5 x 10(3) genome equivalents, each with one copy of nptII) was required to produce one kanamycin-resistant transformant. These experiments and others employing DNA isolated from soil samples demonstrate that the system allows reliable and highly sensitive monitoring of nptII genes in transgenic plant DNA and in DNA from environmental sources, such as soil, without the need for prior DNA amplification (e.g. by PCR).

The nptII(+) gene present in the genome of transgenic potato plants transforms naturally competent cells of the soil bacteria Pseudomonas stutzeri and Acinetobacter BD413 (both harboring a plasmid with an nptII gene containing a small deletion) with the same high efficiency as nptII(+) genes on plasmid DNA (3x10(-5)-1x10(-4) transformants per nptII(+)) despite the presence of a more than 10(6)-fold excess of plant DNA. However, in the absence of homologous sequences in the recipient cells the transformation by nptII(+) dropped by at least about 10(8)-fold in P. stutzeri and 10(9)-fold in Acinetobacter resulting in the latter strain in < or =1x10(-13) transformants per nptII(+). This indicated a very low probability of non-homologous DNA fragments to be integrated by illegitimate recombination events during transformation.

This paper provides an overview of the diversity of tools available for online learning and identifies the drivers of online learning and directives for future research relating to online learning in dentistry. After an introduction and definitions of online learning, this paper considers the democracy of knowledge and tools and systems that have democratized knowledge. It identifies assessment systems and the challenges of online learning. This paper also identifies the drivers for online learning, including those for instructors, administrators and leaders, technology innovators, information and communications technology personnel, global dental associations and government. A consideration of the attitudes of the stakeholders and how they might work together follows, using the example of the unique achievement of the successful collaboration between the Universities of Adelaide, Australia and Sharjah, United Arab Emirates. The importance of the interaction of educational principles and research on online learning is discussed. The paper ends with final reflections and conclusions, advocating readers to move forward in adopting online learning as a solution to the increasing worldwide shortage of clinical academics to teach dental clinicians of the future.