Transformation of Thermus Species by Natural Competence   

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Original research article

A brief version of this protocol appeared in:
Journal of Bacteriology
Jan 2015


Many Thermus species harbour genomes scourged with horizontally transferred signatures. Thermus thermophilus (Tth) has been characterized as naturally competent. The transformation protocol described here is based on the maximum DNA uptake rates registered at mid-exponential phase (OD600 0.3-0.4). Here we describe the stepwise protocol followed for transformation of both plasmids and linearized genomic DNA, of which the latter can be employed as an alternative method to electroporation to introduce mutations or to generate gene deletions in Thermus isolates, for instance.


Thermus thermophilus (Tth) is an extreme thermophilic species extensively used as laboratory model, due to its ancient phylogenetic origin, the comparative ease of crystallisation of its proteins and macromolecular complexes, and the fast growth and good yields under laboratory growth conditions of several of its isolates. Among the most commonly employed isolates, the strains T. thermophilus HB27 and HB8 constitute the most frequently used models due to the high rates by which they can be transformed by natural competence. DNA of any source and topology can be easily taken up by growing cells of these isolates at rates of around 40 kb/s/cell (Schwarzenlander and Averhoff, 2006) showing yields of around 10-2 transformants/viable cell. For this, a polar located natural competence apparatus (Gold et al., 2015) involving at least 16 proteins encoded in five loci in the chromosome (Averhoff, 2009) of both strains is expressed essentially in a constitutive way, although the transformation efficiency is higher at exponential phase. Variants of antibiotic resistance genes encoding thermostable variants have been developed by directed evolution as gene markers, in such a way that selection can be performed in plates with kanamycin (Lasa et al., 1992), hygromycin B (Nakamura et al., 2005), or bleomycin (Brouns et al., 2005). Streptomycin resistance can be also employed to check natural competence with isogenic DNA (Koyama et al., 1986). This protocol describes the highly efficient transformation of cultures at exponential growth phase, providing reproducible data of maximized transformation efficiency.

Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Blesa, A. and Berenguer, J. (2016). Transformation of Thermus Species by Natural Competence. Bio-protocol 6(22): e2007. DOI: 10.21769/BioProtoc.2007.

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