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. Transjugation is a new type of horizontal gene transfer process in which a donor cell pushes out genomic DNA upon cell contact and a recipient cell pulls this DNA inside by natural transformation. Here we describe TdtA, a DNA translocase of the pushing system of T. thermophilus, which is encoded within ICEth1, a new class of Integrative and Conjugative Element whose presence leads to generalized cell-to-cell transfer of any gene marker, circumventing the Argonaute surveillance system that controls access of extracellular DNA acquired by transformation.

Intense gene flux between prokaryotes result in high percentage of archaeal genes in the genome of the thermophilic bacteria Thermus spp. Among these archaeal genes a homolog to the Sulfolobus spp. HerA protein appears in all of the Thermus spp. strains so far sequenced (HepA). The role of HepA in Thermus thermophilus HB27 has been analyzed using deletion mutants, and its structure resolved at low resolution by electron microscopy. Recombinant HepA shows DNA-dependent ATPase activity and its structure revealed a double ring, conically-shaped hexamer with an upper diameter of 150 Å and a bottom module of 95 Å. A central pore was detected in the structure that ranges from 13 Å at one extreme, to 30 Å at the other. Mutants lacking HepA show defective natural competence and DNA donation capability in a conjugation-like process termed “transjugation”, and also high sensitivity to UV and dramatic sensitivity to high temperatures. These data support that acquisition of an ancestral archaeal HerA has been fundamental for the adaptation of Thermus spp. to high temperatures.

Transjugation is an unconventional conjugation mechanism in Thermus thermophilus (Tth) that involves the active participation of both mating partners, encompassing a DNA secretion system (DSS) in the donor and an active natural competence apparatus (NCA) in the recipient cells. DSS is encoded within an integrative and conjugative element (ICETh1) in the strain Tth HB27, whereas the NCA is constitutively expressed in both mates. Previous experiments suggested the presence of multiple origins of transfer along the genome, which could generate genomic mosaicity among the progeny. Here, we designed transjugation experiments between two closely related strains of Tth with highly syntenic genomes, containing enough single nucleotide polymorphisms to allow precise parenthood analysis. Individual clones from the progeny were sequenced, revealing their origin as derivatives of our ICETh1-containing intended “donor” strain (HB27), which had acquired separate fragments from the genome of the ICETh1-free HB8 cells, which are our intended recipient. Due to the bidirectional nature of transjugation, only assays employing competence-defective HB27 derivatives as donors allowed the recovery of HB8-derived progeny. These results show a preference for a retrotransfer mechanism in transjugation in ICETh1-bearing strains, supporting an inter-strain gene-capture function for ICETh1. This function could benefit the donor-capable host by facilitating the acquisition of adaptive traits from external sources, ultimately increasing the open pangenome of Thermus, maximizing the potential repertoire of physiological and phenotypical traits related to adaptation and speciation.

The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3′, 5′-diadenosine monophosphate (c-di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.

Denitrification in Thermus thermophilus is encoded by the nitrate respiration conjugative element (NCE) and nitrite and nitric oxide respiration (nic) gene clusters. A tight coordination of each cluster’s expression is required to maximize anaerobic growth, and to avoid toxicity by intermediates, especially nitric oxides (NO). Here, we study the control of the nitrite reductases (Nir) and NO reductases (Nor) upon horizontal acquisition of the NCE and nic clusters by a formerly aerobic host. Expression of the nic promoters PnirS, PnirJ, and PnorC, depends on the oxygen sensor DnrS and on the DnrT protein, both NCE-encoded. NsrR, a nic-encoded transcription factor with an iron–sulfur cluster, is also involved in Nir and Nor control. Deletion of nsrR decreased PnorC and PnirJ transcription, and activated PnirS under denitrification conditions, exhibiting a dual regulatory role never described before for members of the NsrR family. On the basis of these results, a regulatory hierarchy is proposed, in which under anoxia, there is a pre-activation of the nic promoters by DnrS and DnrT, and then NsrR leads to Nor induction and Nir repression, likely as a second stage of regulation that would require NO detection, thus avoiding accumulation of toxic levels of NO. The whole system appears to work in remarkable coordination to function only when the relevant nitrogen species are present inside the cell.