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. [Int Microbiol 18(3):177-187 (2015)]Keywords: Thermus · horizontal gene transfer · extracellular vesicle

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

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Horizontal gene transfer in Thermus thermophilus: mechanisms and barriers

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 12-02-2016Esta tesis tiene embargado el acceso al texto completo hasta el 12-08-2019La autora no permite ningún tipo de modificación en el excelComparative genome analysis have evidenced that fluent genetic exchange is mainly mediated by Horizontal Gene Transfer (HGT), thus recognized as a leading force of prokaryotic evolution and microbial genetic diversity. Transduction, transformation and conjugation are the three standard mechanisms driving HGT. The ancient thermophilic bacteria Thermus thermophilus laterally transfers DNA in a really efficient way due to a highly sophisticated competence machinery as well as by a conjugation-like process. However, conjugation in T. thermophilus emerges rather unconventional as it is efficient between isogenic cells, thus, bidirectional, and no type IV secretion systems seems to be involved. Besides, genes associated to the megaplasmid are transferred with higher frequencies (~10 fold) than those localized in the chromosome. No evident order in the transfer among megaplasmid markers contrasts with the identification of multiple hotspots of transfer in the chromosome. Several of these loci showing higher transfer frequencies are encoded near putative Tth111 type II restriction sites which might work as OriTs. The competence machinery actively participates in conjugation, being required in the receptor cell but not in the donor. Hence, a two-step model (push-pull) is proposed, where the donor energetically pumps DNA to a receptor cell, which actively pulls in the DNA transferred with its competence apparatus. Two active hexameric ATPases, named CptA and HerA, paralogs to the helicases HerA and Ftsk from other bacteria, have been identified as chief components of the pushing step. Therefore, T. thermophilus is able to proficiently exchange DNA by transformation (including DNA-protected vesicles which could work as long distance vehicles), and largely by conjugation. This fruitful dynamic gene flow is compatible with a battery of protective strategies to prevent potentially harmful invasion of genetic parasites. Among them, the Argonaute protein elicits a DNA-DNA interference on DNA taken up by transformation. We proved larger insights of Argonaute-mediated interference, acting, in vivo, against virtually any kind of DNA template. However, when such DNA is transferred by a conjugation-like process, Argonaute is not activated, suggesting a selective immunity towards the way in which DNA is acquired. This fact, together with the higher efficiency shown by conjugation compared to natural competence when the same DNA was transferred, enforcedthe proposal of conjugation as the major motor of shared traits among populations of Thermus spp in thermal environments. Finally, we analyse the role of the PrimPol polymerase as a ssDNA guide supplier for the Argonaute protein

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-tocell 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.This work has been supported by grant BIO2013-44963-R from the Spanish Ministry of Economy and Competence and FP7-PEOPLE-2012-IAPP grant 324439 from the European Union to J. Berenguer and by grant AV9/6-1 from the Deutsche Forschungsgemeinschaft to B. Averhoff. An institutional grant from the Fundación Ramón Areces to CBMSO and financial support to the Spanish National Network for Extremophilic Microorganisms (BIO2011-12879-E) are also acknowledged.Peer Reviewe

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. © 2014, American Society for Microbiology.Spanish Ministry of Science. An institutional grant from Fundación Ramón ArecesPeer Reviewe

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.post-print1,28 M

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.post-print117 K

A thermostable DNA primase-polymerase from a mobilegenetic element involved in defence againstenvironmental 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.post-print697 K

PUE attack detection in CWSNs using anomaly detection techniques

Cognitive wireless sensor network (CWSN) is a new paradigm, integrating cognitive features in traditional wireless sensor networks (WSNs) to mitigate important problems such as spectrum occupancy. Security in cognitive wireless sensor networks is an important problem since these kinds of networks manage critical applications and data. The specific constraints of WSN make the problem even more critical, and effective solutions have not yet been implemented. Primary user emulation (PUE) attack is the most studied specific attack deriving from new cognitive features. This work discusses a new approach, based on anomaly behavior detection and collaboration, to detect the primary user emulation attack in CWSN scenarios. Two non-parametric algorithms, suitable for low-resource networks like CWSNs, have been used in this work: the cumulative sum and data clustering algorithms. The comparison is based on some characteristics such as detection delay, learning time, scalability, resources, and scenario dependency. The algorithms have been tested using a cognitive simulator that provides important results in this area. Both algorithms have shown to be valid in order to detect PUE attacks, reaching a detection rate of 99% and less than 1% of false positives using collaboration

Controlling the degradation of wireless sensor networks

With the fast expansion of Wireless Sensor Networks (WSNs) and the increasing emergence of new scenarios and applications, extending their lifetime is crucial. Usually, WSN developers use generic algorithms and deployment arrangements without considering the specific needs of their network's application. Taking this application into account can result in a significant enhancement of performance, both in terms of increasing the lifetime and improving the quality of service (QoS). Furthermore, most WSN developers do not consider the final behavior of the network when nodes are nearly depleted and resources are scarce. In this paper we introduce the concept of the controlled degradation of the network, to refer to the strategies aimed at managing this deterioration process. The existing definitions of the network lifetime do not normally consider the specific purpose or application for which the WSN is intended. Thus, they are not suited to describe and test controlled degradation strategies. Consequently, we propose a new formal and comprehensive definition for the network lifetime. Finally, this work presents a proof of concept that confirms our statements and reinforces the potential of this research line