Genetics

Research Projects

Mechanisms of genetic recombination and horizontal gene transfer in bacteria and their contribution to evolution

Bacteria are unique in their ability to exchange genetic material in their habitat. The donor and recipient cells may belong to the same species or to different species or genera. The transferred DNA can become part of the recipient cell genome by recombination catalyzed by bacterial enzymes. Depending on the selective advantage, the newly acquired genetic information may be maintained in a bacterial population or lost. Today it is widely thought that the evolution of bacteria depends besides on mutation, on gene acquisition and gene loss.

The Genetics Group studies horizontal gene transfer between bacteria by naked DNA (natural genetic transformation) and the integration of homologous and foreign DNA by recombination processes. We have recently described a bacterial mechanism for foreign gene acquisition acting by a combination of homologous and non-homologous recombination processes. Now we explore the effectiveness of this mechanism, the enzymes and steps involved and the plasticity of its basic processes (e.g. the interplay between DNA uptake, recombination and mismatch repair). We focus on environmental bacteria and Escherichia coli.

Aspects of applied research including the monitoring of DNA in natural bacterial habitats, safety research on transgenic plants and development of molecular biology tools are also covered by our activities.

Introductions: 

Homologous recombination in Escherichia coli.

The RecBCD enzyme (an enzyme with helicase and nuclease activity) is essential for the initiation of homologous recombination at Chi nucleotide sequences (hot spots of recombination) by producing a recombinogenic 3´ strand for strand transfer by RecA protein. We found that in vivo the enzyme is used up during this process at Chi indicating a stoichiometric role in recombination initiation (1). We also showed that other single-strand specific DNases (incl. exonuclease I, RecJ-exonuclease, SbcCD DNase) are necessary to process duplex DNA ends by trimming off single-stranded tails to make them a substrate for RecBCD catalyzed recombination (2). These trimming steps are also necessary for the recombinational repair of DNA double-strand breaks.

Recent work with multiple single-strand DNase mutants show that single-strand specific DNases have partially overlapping function in recombination with terminally gapped DNA intermediates. However, a quadruple DNase mutant is now found to be recombination deficient for these DNA substrates.

  1. Köppen, A., Krobitsch, S., Thoms, B., Wackernagel, W. (1995) Interaction with the recombination hot spot Chi in vivo converts the RecBCD enzyme of Escherichia coli into a Chi-independent recombinase by inactivation of the RecD subunit. Proc Natl Acad Sci USA 92: 6249-6253.
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  2. Thoms, B., Wackernagel, W. (1998) Interaction of RecBCD enzyme with DNA at double-strand breaks produced in UV-irradiated Escherichia coli: Requirement for DNA end processing. J Bacteriol 180: 5639-5645.
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Recombination and foreign gene acquisition during natural transformation

In 1994 more than 40 bacteria species were known to actively take up DNA from their environment and use it as a source of genetic information (1). We are focussing on two of these, Pseudomonas stutzeri and Acinetobacter, which are ubiquitous in soil and sediment. Previously we have shown that these do not only take up and integrate DNA from their own species but also foreign DNA including DNA from transgenic plants (2).

In particular we study how "foreign" DNA can enter the bacterial genome (3). The novel molecular process termed "homology-facilitated illegitimate recombination" (HFIR) is detailed in the homepage of Dr. Johann de Vries for Acinetobacter sp. (4). Related observations were also made in P. stutzeri ATCC 17587 (5). Foreign gene acquisition by transformation is regulated in P. stutzeri by the mismatch repair system which also affects the HFIR process (6). HFIR can lead to the transfer of DNA from plants to bacteria (7).

  1. Lorenz, M.G., Wackernagel, W. (1994) Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev 58: 563-602.
    [Abstract] [PDF] [Full text] [Reprint]
  2. de Vries, J., Wackernagel, W. (2005) Microbial horizontal gene transfer and the DNA release from transgenic crop plants. Plant Soil, in press.
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  3. de Vries, J., Meier, P., Wackernagel, W. (2001) The natural transformation of the soil bacteria Pseudomonas stutzeri and Acinetobacter sp. by transgenic plant DNA strictly depends on homologous sequences in the recipient cells. FEMS Microbiol Lett 195: 211-215.
    [Abstract] [PDF] [Full text] [Reprint]
  4. de Vries, J., Wackernagel, W. (2002) Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination. Proc Natl Acad Sci USA 99: 2094-2099.
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  5. Meier, P., Wackernagel, W. (2003) Mechanisms of homology-facilitated illegitimate recombination for foreign DNA acquisition in transformable Pseudomonas stutzeri. Mol Microbiol 48: 1107-1118.
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  6. Meier, P., Wackernagel, W. (2005) Impact of mutS inactivation on foreign DNA acquisition by natural transformation in Pseudomonas stutzeri. J Bacteriol 187: 143-154.
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  7. de Vries, J., Herzfeld, T., Wackernagel, W. (2004) Transfer of plastid DNA from tobacco to the soil bacterium Acinetobacter sp. by natural transformation. Mol Microbiol 53: 323-334.
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The DNA uptake by bacteria

Our molecular genetic and physical studies on the DNA uptake during natural transformation have revealed that type IV pili are involved in the transfer of DNA into the periplasm of P. stutzeri. Presently we have identified more than 10 genes necessary for the biogenesis and function of type IV pili (including pilAI, pilB, pilC, pilD, pilAII, pilR, pilT, pilU) and characterized their role in the physical uptake of 3H-labelled DNA (1).

We also identified and characterized further genes which are required for the translocation of DNA from the periplasm through the cytoplasmic membrane incl. comA, exbB, comF, dprA (2).

Moreover, the expression of a pseudopilin gene was found to regulate the level of transformation (3) and the normally necessary function of a molecular motor for DNA uptake can be circumvented by expression of a modified pilAI gene (4).

  1. Graupner, S., Frey, V., Hashemi, R., Lorenz, M.G., Brandes, G., Wackernagel, W. (2000) Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species. J Bacteriol 182: 2184-2190.
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  2. Graupner, S., Wackernagel, W. (2001) Identification and characterization of novel competence genes comA and exbB involved in natural genetic transformation of Pseudomonas stutzeri. Res Microbiol 152: 451-460.
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  3. Graupner, S., Wackernagel, W. (2001) Pseudomonas stutzeri has two closely related pilA genes (type IV pilus structural protein) with opposite influences on natural genetic transformation. J Bacteriol 183: 2359-2366.
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  4. Graupner, S., Weger, N., Sohni, M., Wackernagel, W. (2001) Requirement of novel competence genes pilT and pilU of Pseudomonas stutzeri for natural transformation and suppression of pilT deficiency by a hexahistidine tag on the type IV pilus protein PilAI. J Bacteriol 183: 4694-4701.
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Biomonitoring of recombinant plant DNA in the environment

We have developed bacterial strains as recipients for DNA which allow the highly sensitive and specific monitoring of recombinant DNA (i.e. transgenic plant DNA) by natural transformation. This provides an assay for the functional integrity of DNA that is present in the environment. The assay is similarly sensitive as PCR and does not require in vitro amplification (1, 2, 3). Recent studies show that transgenic plant DNA can persist for up to 5 years in natural nonsterile soil (4).

  1. de Vries, J., Wackernagel, W. (1998) Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation. Mol Gen Genet 257: 606-613.
    [Abstract] [PDF] [Full text] [Reprint]
  2. de Vries, J., Meier, P., Wackernagel, W. (2001) The natural transformation of the soil bacteria Pseudomonas stutzeri and Acinetobacter sp. by transgenic plant DNA strictly depends on homologous sequences in the recipient cells. FEMS Microbiol Lett 195: 211-215.
    [Abstract] [PDF] [Full text] [Reprint]
  3. Meier, P., Wackernagel, W. (2003) Monitoring the spread of recombinant DNA from field plots with transgenic sugar beet plants by PCR and natural transformation of Pseudomonas stutzeri. Transgenic Res 12: 293-304.
    [Abstract] [PDF] [Full text] [Reprint]
  4. de Vries, J., Heine, M., Harms, K., Wackernagel, W. (2003) Spread of recombinant DNA by roots and pollen of transgenic potato plants, identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp. Appl Environ Microbiol 69: 4455-4462.
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Diversification and evolution of bacteria in their habitat

We sample bacteria from their habitat (soil, sediment) and identify members of the same species by molecular methods of which some allow highly sensitive determination of their genetic relatedness. Using such characterized local populations we apply several methods to analyze their structure (such as stochastic methods based on the neutral theory of evolution). Directional selection pressure is the major force in short term evolution, but, in longer terms, turns out to have balancing influence on local bacterial populations by probably cycling between different lineages (1, 4). Local bacterial populations showed high strain diversity (by strong influence of mutation) and complex taxonomic compositions [as a result of differential influence of migrational input (4)]. Also, the identification of barriers to natural transformation between lineages of a bacterial species suggests some similarity to the sexual isolation in eukaryotes which allows free evolutionary development and therefore a step towards speciation (2). Recently, we identified a high heterogeneity of strains of Pseudomonas stutzeri from a geographically very restricted local soil population in their ability for natural transformation (3). The characterization of various isolates from environmental samples increased the number of defined genomovars of P. stutzeri (5).

  1. Sikorski, J., Jahr, H., Wackernagel, W. (2001) The structure of a local population of phytopathogenic Pseudomonas brassicacearum from agricultural soil indicates development under purifying selection pressure. Environ Microbiol 3: 176-186.
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  2. Lorenz, M.G., Sikorski, J. (2000) The potential for intraspecific horizontal gene exchange by natural genetic transformation: sexual isolation among genomovars of Pseudomonas stutzeri. Microbiology 146: 3071-3080.
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  3. Sikorski, J., Teschner, N. Wackernagel, W. (2002) Highly different levels of natural transformation are associated with genomic subgroups within a local population of Pseudomonas stutzeri from soil. Appl Environ Microbiol 68: 865-873.
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  4. Sikorski, J., Möhle, M., Wackernagel, W. (2002) Identification of complex composition, strong strain diversity and directional selection in local Pseudomonas stutzeri populations from marine sediment and soils. Environ Microbiol 4: 465-476.
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  5. Sikorski, J., Lalucat, J., Wackernagel, W. (2005) Genomovars 11 to 18 of Pseudomonas stutzeri, identified among isolates from soil and marine sediment. IJSEM, submitted.
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