Experimental methods for assaying natural transformation and inferring horizontal gene transfer.

The observation of frequent lateral acquisitions of genes in sequenced bacterial genomes has spurred experimental investigations to elucidate the factors governing ongoing gene transfer processes in bacteria. The uptake of naked DNA by natural transformation is known to occur in a wide range of bacterial species and in some archaea. We describe a series of protocols designed to dissect the natural genetic transformability of individual bacterial strains under conditions that progress from standard in vitro conditions to purely in situ, or natural, conditions. One of the most important factors in ensuring the success of any transformation assay system is the use of a sensitive, effective, and distinguishable selection regimen. Detailed template protocols for assaying bacterial transformation in vitro are presented using the naturally competent bacterium Acinetobacter baylyi strain BD413 as a model. Factors increasing the complexity of the assay systems are included in the following section describing the incorporation of components of natural systems to the in vitro models, such as in soil and water microcosm experiments. We then present template protocols for the transformation of bacteria in modified natural systems, such as in the presence of host tissues and extracts or in the greenhouse. Clear and ecologically meaningful demonstrations of in situ natural transformation are most desirable but are also the most complex and challenging. Because of the highly variable nature of these experiments, we include a discussion of important factors that should be considered when designing such experiments. Some advantages and disadvantages of the experimental systems with regard to resolving the hypotheses tested are included in each section.