Abstract
The ability to transfer DNA via direct cell-to-cell contact-dependent process similar to conjugation has been described in Thermus thermophilus (Tth). Here, we detail the mating experiment protocol involving the lateral transfer of thermostable antibiotic resistance markers (i.e., kanamycin: KmR; hygromycin: HygR) between Thermus cells, enabling the selection and quantification of the transfer frequencies. Briefly, liquid cultures of both mates are mixed and laid onto a nitrocellulose filter on a TB plate. After incubation at 60 °C, filters are resuspended upon selective plating. The contribution of DNA uptake by transformation is abolished by the addition of DNase I to the mix. This protocol can be used for the transfer of large DNA fragments (> 10 kb) to Thermus species.
Background
Conjugation, as the chief mechanism for horizontal gene transfer for most bacteria, is a highly specialized process by which DNA is transferred between two cells which are in direct contact (Lederberg and Tatum, 1946). Classical conjugation involves the unidirectional transfer of a DNA molecule, generally plasmid-encoded, from a donor to a recipient cell, which remains passive. However, alternative models have been described for a wide variety of bacteria. In the laboratory, conjugation experiments are fruitful for marker-exchange mutagenesis, among others. Over other methods of genetic transfer, conjugation is advantageous in terms of minimal disruption of the bacterial envelope and the feasibility to transfer large DNA fragments, including large chromosomal regions (> 10 kb). A conjugation-like process among T. thermophilus cells was reported a decade ago, where chromosomal markers were transferred following a high frequency of recombinants Hfr-like process, as evidenced by interrupted mating assays employing liquid mixes of different strains (Ramírez-Arcos et al., 1998). Recently, the model proposed for Thermus thermophilus describes the transfer as a two-step bidirectional process where a functional competence apparatus is required in the recipient cell but not in the donor, bestowing this phenomenon the name of ‘transjugation’ (Blesa et al., 2015). Compared to liquid mating assays aforementioned, transfer frequencies obtained with the protocol described here are higher and more robust. Higher reproducibility of the assays is reached with this protocol compared to mating tests in liquid. Besides, validation of the conjugative transfer is ensured by the addition of DNase.
Materials and Reagents
Notes:
Equipment
Procedure
A schematic summary is also provided underneath (Figure 2).
Data analysis
Notes
Recipes
*Note: The analytical composition of the spring mineral water employed, per mg/ml, is listed beneath (Table 1). Other mineral waters easily purchasable such as Evian water or a laboratory solution resembling this recipe, can be employed. Table 1. Mineral composition of the spring water from Jaraba Spring in Zaragoza (Spain)
Acknowledgments
This work has been funded by Grants BIO2013-44963-R, and FP7-PEOPLE-2012-IAPP No. 324439. A. Blesa held an FPI contract from the Spanish Ministry of Economy and Competitivity.
References
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