The PulE ATPase is required for twitching motility and DNA donation during Thermus thermophilus transjugation.

Thermus thermophilus can acquire DNA through natural competence and through transjugation, a mechanism that involves a two-step process of DNA secretion (push) and DNA internalization (pull) between mating cells of related species. The natural competence apparatus (NCA) is required in the recipient mate for the pull step. However, how the DNA exits of the donor cell is only partially known. The putative DNA translocase TdtA, encoded in mobile genetic element ICETh1 of T. thermophilus HB27, was shown to be required for DNA donation as reported by (Blesa et al. 2017a). This ring-shaped hexameric ATPase binds to the membrane and likely interacts with yet unknown secretory components that allow the extrusion of DNA through the membrane; thus, a genetic screening to identify additional putative secretory components was performed. Here, we describe that mutants in gene TT_C1844, which encodes a putative AAA-ATPase named PulE, do not synthesize the recently described "narrow" type 4 pili required for twitching motility and made up of the major PilA5 pilin. Concomitantly, pulE mutants also exhibited DNA donation defects during transjugation, suggesting a role of narrow pili in the donation process. However, single pilA5 null mutants still function as DNA donors in transjugation experiments, so we conclude that the need for PulE in transjugation is independent of its role in narrow pili synthesis and twitching motility.

ICETh1 and ICETh2, two interdependent mobile genetic elements in Thermus thermophilus transjugation.

Cell to cell DNA transfer between Thermus thermophilus, or transjugation, requires the natural competence apparatus (NCA) of the recipient cell and a DNA donation machinery in the donor. In T. thermophilus HB27, two mobile genetic elements with functional similarities to Integrative and Conjugative Elements (ICEs) coexist, ICETh1 encoding the DNA transfer apparatus and ICETh2, encoding a putative replication module. Here, we demonstrate that excision and integration of both elements depend on a single tyrosine recombinase encoded by ICETh2, and that excision is not required but improves the transfer of these elements to a recipient cell. These findings along with previous results suggest that ICETh1 and ICETh2 depend on each other for spreading among T. thermophilus by transjugation.

A thermostable DNA primase-polymerase from a mobile genetic element involved in defence against environmental DNA.

Primase-polymerases (Ppol) are one of the few enzymes able to start DNA synthesis on ssDNA templates. The role of Thermus thermophilus HB27 Ppol, encoded along a putative helicase (Hel) within a mobile genetic element (ICETh2), has been studied. A mutant lacking Ppol showed no effects on the replication of the element. Also, no apparent differences in the sensitivity to DNA damaging agents and other stressors or morphological changes in the mutant cells were detected. However, the mutants lacking Ppol showed an increase in two to three orders of magnitude in their transformation efficiency with plasmids and genomic DNA acquired from the environment (eDNA), independently of its origin and G + C content. In contrast, no significant differences with the wild type were detected when the cells received the DNA from other T. thermophilus partners in conjugation-like mating experiments. The similarities of this behaviour with that shown by mutants lacking the Argonaute (ThAgo) protein suggests a putative partnership Ppol-ThAgo in the DNA-DNA interference mechanism of defence, although other eDNA defence mechanisms independent of ThAgo cannot be discarded.

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.

Horizontal Gene Transfer in Thermus spp.

The small amount of genetic content in thermophiles generally limits their adaptability to environmental changes. In Thermus spp., very active horizontal gene transfer (HGT) mechanisms allow the rapid spread of strain-specific adaptive gene modules among the entire population. Constitutive expression of a rather particular and highly efficient DNA transport apparatus (DTA) is at the center of this HGT-mediated enhanced adaptability. The function of the DTA is dependent on the integrity and longevity of the extracellular DNA (eDNA) being transformed, which can be improved by the production of extracellular vesicles (EV) through lysis of a fraction of the population. The DTA must also contend with the recipient cell's defensive barriers, namely restriction enzymes, a panoply of CRISPR-Cas systems, and the argonaute-like protein TtAgo, which may be bypassed by transjugation, a new class of bidirectional transformation-dependent conjugation. Efficient transjugation depends on the presence of the ICETh1, an integrative and conjugative element which promotes simultaneous, generalized DNA transfer from several points in the genome. Transjugation shows preference for genes located within a megaplasmid replicon, where the main strain-specific adaptive modules are located. Contribution of transformation, vesicle-mediated eDNAs, and transjugation to HGT in this genus is discussed.

Characterization of a promiscuous cadmium and arsenic resistance mechanism in Thermus thermophilus HB27 and potential application of a novel bioreporter system.

BACKGROUND: The characterization of the molecular determinants of metal resistance has potential biotechnological application in biosensing and bioremediation. In this context, the bacterium Thermus thermophilus HB27 is a metal tolerant thermophile containing a set of genes involved in arsenic resistance which, differently from other microbes, are not organized into a single operon. They encode the proteins: arsenate reductase, TtArsC, arsenic efflux membrane transporter, TtArsX, and transcriptional repressor, TtSmtB. RESULTS: In this work we show that the arsenic efflux protein TtArsX and the arsenic responsive transcriptional repressor TtSmtB are required to provide resistance to cadmium. We analyzed the sensitivity to Cd(II) of mutants lacking TtArsX, finding that they are more sensitive to this metal than the wild type strain. In addition, using promoter probe reporter plasmids, we show that the transcription of TtarsX is also stimulated by the presence of Cd(II) in a TtSmtB-dependent way. Actually, a regulatory circuit composed of TtSmtB and a reporter gene expressed from the TtarsX promoter responds to variation in Cd(II), As(III) and As(V) concentrations. CONCLUSIONS: Our results demonstrate that the system composed by TtSmtB and TtArsX is responsible for both the arsenic and cadmium resistance in T. thermophilus. The data also support the use of T. thermophilus as a suitable chassis for the design and development of As-Cd biosensors.

Noncanonical cell-to-cell DNA transfer in Thermus spp. is insensitive to argonaute-mediated interference.

Horizontal gene transfer drives the rapid evolution of bacterial populations. Classical processes that promote the lateral flow of genetic information are conserved throughout the prokaryotic world. However, some species have nonconserved transfer mechanisms that are not well known. This is the case for the ancient extreme thermophile Thermus thermophilus. In this work, we show that T. thermophilus strains are capable of exchanging large DNA fragments by a novel mechanism that requires cell-to-cell contacts and employs components of the natural transformation machinery. This process facilitates the bidirectional transfer of virtually any DNA locus but favors by 10-fold loci found in the megaplasmid over those in the chromosome. In contrast to naked DNA acquisition by transformation, the system does not activate the recently described DNA-DNA interference mechanism mediated by the prokaryotic Argonaute protein, thus allowing the organism to distinguish between DNA transferred from a mate and exogenous DNA acquired from unknown hosts. This Argonaute-mediated discrimination may be tentatively viewed as a strategy for safe sharing of potentially "useful" traits by the components of a given population of Thermus spp. without increasing the genome sizes of its individuals.

Wastewater treatment plant effluents change abundance and composition of ammonia-oxidizing microorganisms in mediterranean urban stream biofilms.

Streams affected by wastewater treatment plant (WWTP) effluents are hotspots of nitrification. We analyzed the influence of WWTP inputs on the abundance, distribution, and composition of epilithic ammonia-oxidizing (AO) assemblages in five Mediterranean urban streams by qPCR and amoA gene cloning and sequencing of both archaea (AOA) and bacteria (AOB). The effluents significantly modified stream chemical parameters, and changes in longitudinal profiles of both NH(4)(+) and NO(3)(-) indicated stimulated nitrification activity. WWTP effluents were an allocthonous source of both AOA, essentially from the Nitrosotalea cluster, and mostly of AOB, mainly Nitrosomonas oligotropha, Nitrosomonas communis, and Nitrosospira spp. changing the relative abundance and the natural composition of AO assemblages. Under natural conditions, Nitrososphaera and Nitrosopumilus AOA dominated AO assemblages, and AOB were barely detected. After the WWTP perturbation, epilithic AOB increased by orders of magnitude whereas AOA did not show quantitative changes but a shift in population composition to dominance of Nitrosotalea spp. The foraneous AOB successfully settled in downstream biofilms and probably carried out most of the nitrification activity. Nitrosotalea were only observed downstream and only in biofilms exposed to either darkness or low irradiance. In addition to other potential environmental limitations for AOA distribution, this result suggests in situ photosensitivity as previously reported for Nitrosotalea under laboratory conditions.

Transferable denitrification capability of Thermus thermophilus.

Laboratory-adapted strains of Thermus spp. have been shown to require oxygen for growth, including the model strains T. thermophilus HB27 and HB8. In contrast, many isolates of this species that have not been intensively grown under laboratory conditions keep the capability to grow anaerobically with one or more electron acceptors. The use of nitrogen oxides, especially nitrate, as electron acceptors is one of the most widespread capabilities among these facultative strains. In this process, nitrate is reduced to nitrite by a reductase (Nar) that also functions as electron transporter toward nitrite and nitric oxide reductases when nitrate is scarce, effectively replacing respiratory complex III. In many T. thermophilus denitrificant strains, most electrons for Nar are provided by a new class of NADH dehydrogenase (Nrc). The ability to reduce nitrite to NO and subsequently to N2O by the corresponding Nir and Nor reductases is also strain specific. The genes encoding the capabilities for nitrate (nar) and nitrite (nir and nor) respiration are easily transferred between T. thermophilus strains by natural competence or by a conjugation-like process and may be easily lost upon continuous growth under aerobic conditions. The reason for this instability is apparently related to the fact that these metabolic capabilities are encoded in gene cluster islands, which are delimited by insertion sequences and integrated within highly variable regions of easily transferable extrachromosomal elements. Together with the chromosomal genes, these plasmid-associated genetic islands constitute the extended pangenome of T. thermophilus that provides this species with an enhanced capability to adapt to changing environments.

Methods to Identify and Analyze Vesicle-Protected DNA Transfer.

Conjugation, transformation, and transduction constitute the three classical mechanisms involved in horizontal gene transfer (HGT) among prokaryotes. In addition, alternative HGT mechanisms exist in groups of organisms. Among them, the use of DNA-containing membrane vesicles as shuttle elements for HGT has been described for a number of microorganisms, including both thermophiles and mesophiles. Here we describe the methods followed to detect, purify, and analyze these vesicles.

Nitrate Respiration in Thermus thermophilus NAR1: from Horizontal Gene Transfer to Internal Evolution.

Genes coding for enzymes of the denitrification pathway appear randomly distributed among isolates of the ancestral genus Thermus, but only in few strains of the species Thermus thermophilus has the pathway been studied to a certain detail. Here, we review the enzymes involved in this pathway present in T. thermophilus NAR1, a strain extensively employed as a model for nitrate respiration, in the light of its full sequence recently assembled through a combination of PacBio and Illumina technologies in order to counteract the systematic errors introduced by the former technique. The genome of this strain is divided in four replicons, a chromosome of 2,021,843 bp, two megaplasmids of 370,865 and 77,135 bp and a small plasmid of 9799 pb. Nitrate respiration is encoded in the largest megaplasmid, pTTHNP4, within a region that includes operons for O2 and nitrate sensory systems, a nitrate reductase, nitrate and nitrite transporters and a nitrate specific NADH dehydrogenase, in addition to multiple insertion sequences (IS), suggesting its mobility-prone nature. Despite nitrite is the final product of nitrate respiration in this strain, the megaplasmid encodes two putative nitrite reductases of the cd1 and Cu-containing types, apparently inactivated by IS. No nitric oxide reductase genes have been found within this region, although the NorR sensory gene, needed for its expression, is found near the inactive nitrite respiration system. These data clearly support that partial denitrification in this strain is the consequence of recent deletions and IS insertions in genes involved in nitrite respiration. Based on these data, the capability of this strain to transfer or acquire denitrification clusters by horizontal gene transfer is discussed.

Integrative and Conjugative Element ICETh1 Functions as a Pangenomic DNA Capture Module in Thermus thermophilus.

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.

Complete Genome Sequence of Mycolicibacterium hassiacum DSM 44199.

Mycolicibacterium hassiacum is the most thermophilic of all the mycobacteria. A partial sequence based on Illumina technology of around 5 Mbp was published in 2012. Here, we report the 5,269,097-bp complete genome sequence assembled into a single circular chromosome.

Into the Thermus Mobilome: Presence, Diversity and Recent Activities of Insertion Sequences Across Thermus spp.

A high level of transposon-mediated genome rearrangement is a common trait among microorganisms isolated from thermal environments, probably contributing to the extraordinary genomic plasticity and horizontal gene transfer (HGT) observed in these habitats. In this work, active and inactive insertion sequences (ISs) spanning the sequenced members of the genus Thermus were characterized, with special emphasis on three T. thermophilus strains: HB27, HB8, and NAR1. A large number of full ISs and fragments derived from different IS families were found, concentrating within megaplasmids present in most isolates. Potentially active ISs were identified through analysis of transposase integrity, and domestication-related transposition events of ISTth7 were identified in laboratory-adapted HB27 derivatives. Many partial copies of ISs appeared throughout the genome, which may serve as specific targets for homologous recombination contributing to genome rearrangement. Moreover, recruitment of IS1000 32 bp segments as spacers for CRISPR sequence was identified, pointing to the adaptability of these elements in the biology of these thermophiles. Further knowledge about the activity and functional diversity of ISs in this genus may contribute to the generation of engineered transposons as new genetic tools, and enrich our understanding of the outstanding plasticity shown by these thermophiles.

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.

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

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.

An ArsR/SmtB family member regulates arsenic resistance genes unusually arranged in Thermus thermophilus HB27.

Arsenic resistance is commonly clustered in ars operons in bacteria; main ars operon components encode an arsenate reductase, a membrane extrusion protein, and an As-sensitive transcription factor. In the As-resistant thermophile Thermus thermophilus HB27, genes encoding homologues of these proteins are interspersed in the chromosome. In this article, we show that two adjacent genes, TtsmtB, encoding an ArsR/SmtB transcriptional repressor and, TTC0354, encoding a Zn2+ /Cd2+ -dependent membrane ATPase are involved in As resistance; differently from characterized ars operons, the two genes are transcribed from dedicated promoters upstream of their respective genes, whose expression is differentially regulated at transcriptional level. Mutants defective in TtsmtB or TTC0354 are more sensitive to As than the wild type, proving their role in arsenic resistance. Recombinant dimeric TtSmtB binds in vitro to both promoters, but its binding capability decreases upon interaction with arsenate and, less efficiently, with arsenite. In vivo and in vitro experiments also demonstrate that the arsenate reductase (TtArsC) is subjected to regulation by TtSmtB. We propose a model for the regulation of As resistance in T. thermophilus in which TtSmtB is the arsenate sensor responsible for the induction of TtArsC which generates arsenite exported by TTC0354 efflux protein to detoxify cells.

Contribution of vesicle-protected extracellular DNA to horizontal gene transfer in Thermus spp.

Highly efficient apparatus for natural competence and conjugation have been shown as the major contributors to horizontal gene transfer (HGT) in Thermus thermophilus. In practical terms, both mechanisms can be distinguished by the sensitivity of the former to the presence of DNAse, and the requirement for cell to cell contacts in the second. Here we demonstrate that culture supernatants of different strains of Thermus spp. produce DNAse-resistant extracellular DNA (eDNA) in a growth-rate dependent manner. This eDNA was double stranded, similar in size to isolated genomic DNA (around 20 kbp), and represented the whole genome of the producer strain. Protection against DNAse was the consequence of association of the eDNA to membrane vesicles which composition was shown to include a great diversity of cell envelope proteins with minor content of cytoplasmic proteins. Access of the recipient cell to the protected eDNA depended on the natural competence apparatus and elicited the DNA-DNA interference defence mediated by the Argonaute protein. We hypothesize on the lytic origin of the eDNA carrying vesicles and discuss the relevance of this alternative mechanism for HGT in natural thermal environments.