Hierarchical control of nitrite respiration by transcription factors encoded within mobile gene clusters of Thermus thermophilus

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

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

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.Spanish Ministry of Economy and Competitiveness (BIO2013-44963-R and BIO2016-77031-R to JB and BFU2014-55475-R to JRC), the FP7-PEOPLE-2012-IAPP from the European Union (Grant number 324439 to JB), and the Comunidad AutoÂnoma de Madrid (S2013/MIT-2807 to JRC). An institutional grant from Fundación Ramón Areces to CBMSOPeer Reviewe

TdtA is required in the donor strain for transjugation.

<p><b>(A)</b> SDS-PAGE electrophoresis showing membrane protein profiles. Patterns corresponding to the <i>ΔtdtA</i>::<i>kat</i> mutants derived from NAR1 (N) and HB27 (H) and the corresponding patterns after transjugation between NAR1-<i>ΔtdtA</i> and HB27-Wt (T1) and between HB27-<i>ΔtdtA</i> and NAR1-Wt (T2). Lane M shows protein molecular standards at 97.4, 66.2, 45, 31, and 21.5 kDa. Large and small arrowheads signal the S layer proteins of 100 kDa and 97 kDa corresponding to the NAR1 and HB27 strains, respectively, used as main strain identification marker. Note the similarities between lanes N and T1, and between lanes H and T2, supporting that transjugants derive from the respective <i>tdtA</i> mutant in the matings. <b>(B)</b> Agarose gel electrophoresis showing PCR amplicons of <i>nrcE</i> (upper panel), specific to the NAR1 strain, and <i>ttp0220</i> (lower panel), specific to the HB27 strain using DNA extracted from <i>ΔtdtA</i>::<i>kat</i> mutants derived from NAR1 (N) and HB27 (H), the transjugants pool from mating experiments between NAR1-<i>ΔtdtA</i> and HB27-Wt (T1), and the transjugants pool of the reciprocal mating between HB27-<i>ΔtdtA</i> and NAR1-Wt (T2). Oligonucleotide sequences are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006669#pgen.1006669.s004" target="_blank">S2 Table</a>.</p

Phenotypic effects of deletion of FtsK-HerA homologs encoded by <i>T</i>. <i>thermophilus</i> HB27.

<p>Phenotypic effects of deletion of FtsK-HerA homologs encoded by <i>T</i>. <i>thermophilus</i> HB27.</p

Expression and subcellular localization of TdtA.

<p><b>(A)</b> The expression of the TdtA-YFP fusion from its native promoter in the chromosome was followed by western blot with anti-GFP antiserum throughout growth at 60°C. Identical cell mass was analyzed at the indicated optical densities at 550 nm <b>(B)</b> Western blot with an antiserum that cross-reacts with both TdtA and a HerA-like protein (product of TTC0147 baptized as HepA) was used to localize the proteins in soluble (S) and non-soluble (P) fractions from the following strains of <i>T</i>. <i>thermophilus</i> HB27: wild type (Wt), Δ<i>hepA</i>::<i>kat</i> (<i>hepA</i>), Δ<i>tdtA</i>::<i>kat</i> (<i>tdtA</i>) and Δ<i>tdtA</i>::<i>kat</i>, <i>hepA</i>::<i>hyg</i> (<i>hepA</i>,<i>tdtA</i>). The proteins detected in each case are indicated underneath: TdtA (T) and HepA (H).</p

The product of TTC1879 (TdtA) is required for transjugation.

<p>Transfer frequencies are expressed as the ratio of transjugant: wild type CFU. Bars 1 and 2 correspond to matings between a <i>tdtA</i> mutant (<i>ΔtdtA</i>) and a Hyg^r wild type strain (wt^H) (1) or a <i>ΔpilA4</i> competence mutant (<i>Δpil</i>) (2). Mating between the wt^H strain and a double <i>tdtA</i> and <i>ΔpilA4</i> mutant (<i>ΔtdtA-ΔpilA)</i> rendered no transjugants (3). The expression of TdtA from a plasmid in this double mutant (<i>Δpil-ΔtdtA</i>^<i>comp</i>) allowed for the generation of transjugants in matings with a Cm^r wild type (wt^Cm) (4). Control matings between the same wt^Cm strain and a single <i>ΔpilA4</i> mutant carrying the empty plasmid (<i>Δpil/pMH</i>) showed a 10-fold higher transjugation frequency (5). ANOVA tests showed significant differences among frequencies of transfer of all the derivatives plotted (<i>p</i>-value< 0.001) and <i>post-hoc</i> Holm Sidak tests proved that absence of <i>tdtA</i> has an effect on transjugation (n = 8). Asterisks indicate significant statistical differences compared to the wild type (*: p-value>0.05;**<i>p</i>-value<0.001).</p

Effects of the NurA-homolog and Tth111II on transjugation.

<p>The frequencies of transjugation assays between <i>pilA</i> mutants labeled with kanamycin at the <i>gdh</i> (<i>wt</i>,<i>pil</i>) locus (1), or at the genes encoding Tth111II (<i>tth</i>,<i>pil</i>) (2), NurA-like (<i>nurA</i>,<i>pil</i>) (3) or TdtA (<i>tdtA</i>,<i>pilA</i>) (4) and a wild type strain labeled with Hyg^r are shown. Asterisks indicate significant statistical differences compared to the wild type (<i>p</i>-value<0.001) (n = 6).</p

Bacterial strains used in this work.

<p>Bacterial strains used in this work.</p

Transjugation is associated with presence of ICEth1.

<p>Scheme of the experimental design followed to unequivocally associate ICEth1 and transjugation efficiency. <b>(A)</b> ICEth1 (red dot) was labeled with Km^r (<i>ICEth1</i>::<i>pK</i>) in a HB27Δ<i>pilA4</i> background and transjugated into an HB8 strain labeled with Hyg^r (orange triangle), which naturally lacks this element. <b>(B)</b> The HB8 containing ICEth1 (Hyg^r, Km^r) was mated with a Cm^r derivative of HB8, using a Hyg^r, <i>pyrE</i>::<i>pK</i> (blue triangle) strain as a control. <b>(C)</b> Transfer frequencies detected for the matings described in B above. Frequencies of the ICEth1-containing strain are the average value from 7 donor clones in three independent experiments. A similar number of assays were carried out for the strain lacking ICEth1.</p

TdtA single-particle electron microscopy reconstruction.

<p><b>(A)</b> Representative electron micrograph of a negatively stained TdtA sample; bar = 50 nm. Six two-dimensional averaged classes of the oligomeric TdtA are shown (right). <b>(B)</b> Three-dimensional reconstruction of the hexameric TdtA. <b>(C)</b> Semitransparent model of the hexameric TdtA with the fitted atomic model of the hexameric HerA from <i>S</i>. <i>solfataricus</i> (pink). Arrows indicate the HerA region (residues 216–289), which remains outside the TdtA ring.</p