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Section 5 - Detection of gene transfer in the environment

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Molecular Microbial Ecology Manual

Natural transformation in aquatic environments

Introduction

Transformation is a process in which competent cells take up DNA and incorporate it into their genome. It is one of the mechanisms by which genes may be spread through natural populations [ 27]. A wide variety of bacteria express competence (the physiological ability to take up DNA) during normal growth. Frischer et al. [ 9] found up to 16% of isolates taken from Tampa Bay were naturally competent. When competent cells come into contact with a source of DNA, this is bound to the cell and taken up. As well as DNA in free solution, competent bacteria can take up DNA associated with particulate matter [ 15, 16, 29], cellular debris [ 14], heat inactivated cells [ 23, 30] and intact, live donor cells [ 1, 23, 24, 28].

Transformation is generally most efficient with homologous chromosomal DNA. Once inside the cell it can be integrated into the host's chromosome by normal recombination processes [ 5, 7]. However, in many cases...

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Notes

  1. 1.

    The sediment to be used can be autoclaved or non-sterile, depending on the purpose of the study. We have only observed transfer in autoclaved sediments. If non-sterile sediments are to be used, there will be a high level of resistance to kanamycin and streptomycin amongst the indigenous flora. Hopefully, colony hybridization would identify transformants.

  2. 2.

    There is usually a background level of resistance to kanamycin and streptomycin in most marine microbial communities. Therefore, it is imperative to verify plasmid acquisition, both by colony hybridization of controls and treatment plates and also by selecting individual hybridizing colonies for further study. Miniprep, restriction analysis, and Southern hybridization of these clones may still yield equivocal results. Restriction profiles of pQSR50 are often changed, either from marine bacterial methylation systems or by plasmid rearrangement [ 10].

  3. 3.

    Again, seawater to be used can be nonsterile, sterile filtered, or autoclaved, sterile filtered. The volumes can be scaled up and the experiments performed in situ using Fenwall Gas Permeable Tissue Culture Bags (Fenwall Scientific). It may be difficult to enumerate the donor population on LB containing kanamycin and streptomycin because of the high level of indigenous resistant organisms found in some environments. However, in our experience, there are usually no indigenous organisms that can grow on the KSNR plates.

  4. 4.

    For strain NF1815/RP4 use: 5 ml LB + tetracycline (Tc) - 20 μg/ml at 37 °C with 200 rpm agitation.

  5. 5.

    Use appropriate antibiotics if other bacterial strains or plasmids are being used.

  6. 6.

    Sand is inoculated in batch. One batch is inoculated with both donor and recipient and two additional ones are inoculated only with donor and only with recipient (controls) (see Fig. 4).

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  1. Department of Marine Science, University of South Florida, St Petersburg Campus, 140 Seventh Avenue South, St. Petersburg, Fl 33701, USA

    John H. Paul & Haydn G. Williams

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G. A. Kowalchuk F. J. de Bruijn I. M. Head A. D. Akkermans J. D. van Elsas

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Paul, J.H., Williams, H.G. (2004). Section 5 - Detection of gene transfer in the environment. In: Kowalchuk, G.A., de Bruijn, F.J., Head, I.M., Akkermans, A.D., van Elsas, J.D. (eds) Molecular Microbial Ecology Manual. Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-2177-0_5

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