The transjugation machinery of Thermus thermophilus: Identification of TdtA, an ATPase involved in DNA donation

Abstract

In addition to natural competence, some Thermus thermophilus strains show a high rate of DNA transfer via direct cell-to-cell contact. The process is bidirectional and follows a two-step model where the donor cell actively pushes out DNA and the recipient cell employs the natural competence system to take up the DNA, in a hybrid transformation-dependent conjugation process (transjugation). While the DNA uptake machinery is well known as in other bacterial species that undergo transformation, the pushing step of transjugation remains to be characterized. Here we have searched for hypothetical DNA translocases putatively involved in the pushing step of transjugation. Among candidates encoded by T. thermophilus HB27, the TdtA protein was found to be required for DNA pushing but not for DNA pulling during transjugation, without affecting other cellular processes. Purified TdtA shows ATPase activity and oligomerizes as hexamers with a central opening that can accommodate double-stranded DNA. The tdtA gene was found to belong to a mobile 14 kbp-long DNA element inserted within the 3′ end of a tRNA gene, flanked by 47 bp direct repeats. The insertion also encoded a homolog of bacteriophage site-specific recombinases and actively self-excised from the chromosome at high frequency to form an apparently non-replicative circular form. The insertion also encoded a type II restriction endonuclease and a NurA-like nuclease, whose activities were required for efficient transjugation. All these data support that TdtA belongs to a new type of Integrative and Conjugative Element which promotes the generalized and efficient transfer of genetic traits that could facilitate its co-selection among bacterial populations.

Horizontal Gene Transfer in Thermus spp.

“…Still more surprising was the requirement of a fully active natural competence system in the recipient cells, in such a way that mutants deficient in virtually any described component of the DTA could serve only as DNA donor, a property that led to proposal of the term transjugation (transformation-dependent conjugation) (Blesa et al, 2017). Further parenthood analysis experiments, in which combinations of non-competent mutants were used, confirmed that the DNA-donation machinery was independent of the competence apparatus, both present in the same bacteria, implying a two-step ("push and pull") model for transjugation.…”

“…The search for putative components of the DNApushing system led to the identification of transjugation donor translocase A (TdtA), a protein belonging to the FtsK-HerA family of DNA translocase-helicases. Its mutation impaired the ability of the cell to act as a donor in transjugation, without significant effect on natural competence or any other phenotypic trait (Blesa et al, 2017). Purified recombinant TdtA was subsequently shown to form hexameric rings in the presence of ATP, with a central pore wide enough to accommodate dsDNA, resembling the TraB protein encoded by conjugative plasmids of Streptomyces spp.…”

“…Interestingly, the TdtA protein is only present in a few of the sequenced strains of T. thermophilus, encoded by a mobile genetic element (ICETh1) reminiscent of the integrative and conjugative elements (ICE) found in many conjugative bacteria (Blesa et al, 2017). This 14 kb element encodes a typical phage-like recombinase of the XerC family and a four-gene operon encoding: a type IIG restriction endonuclease identical in sequence to Tth111II (Zhu et al, 2014); a putative nuclease of the NurA family; TdtA; and a putative DNA methylase.…”

“…The main HGT mechanisms in T. thermophilus involve a DTA that can act directly on free eDNA (Averhoff, 2009) as described in other bacteria, or uptake eDNA from extracellular vesicles (EV) (Blesa and Berenguer, 2015), or directly transfer from donor ! 24 cells in intimate contact in a process called transjugation (Blesa et al, 2017).…”

Role of Archaeal HerA Protein in the Biology of the Bacterium Thermus thermophilus

“…Inferential and comparative assays were performed when necessary and include Student’s t -tests, Kruskal-Wallis one-way analysis, Wilcoxon tests, and one-way analysis of variance (ANOVA) test, as described [15]. …”