Biotechnology and genetics of extreme thermophiles

Research summary:

Our group uses thermophiles as models for the study of ancestral biological processes and as source of thermostable enzymes and selection platforms for the development of new tools for biotechnological applications. Our main laboratory model is Thermus thermophilus an extreme thermophile bacterium that is easy to grow under laboratory conditions and amenable to genetic manipulation due to the presence of a highly efficient natural competence apparatus (NCA).

The main biological topics under study in our group during the last years are related with new and unconventional conjugative mechanisms of horizontal gene transfer among Thermus spp that generate genomic mosaicism in the progeny as consequence of the  simultaneous transfer of DNA from several points of the genome of the donor to the recipient cell. The transfer capacity is encoded by small complementary integrative and conjugative elements whose biology is also under study. Further, barriers based on DNA-DNA interference to horizontal gene transfer mediated by argonaute-like proteins are also a focus of our interest.

The group is also interested in new biotechnological applications derived from the use of thermophiles. A major effort focuses on the discovery of thermostable enzymes and in the isolation of thermostable variants of enzymes that could better respond to the requirements for industrial biocatalysts or in other applications in the field of Molecular Biology, such as gene edition. For this, new multifunctional vectors are being developed which allow a more efficient identification of thermostable enzymes from metagenomic libraries or the thermo-selection of variants of unstable enzymes of biotechnological interest in modified thermophile host strains.

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Figure 1: Extracellular DNA that enters Tth by the NCA faces its degradation by ThAgo through gDNA-DNA interference. In contrast, dsDNA transferred by a donor Tth cell through the TdtA translocase and incorporated through the NCA is not susceptible to ThAgo. The DNA helicase HepA is also required in the donor, likely to repair the scars generated in the genome in the almost random donation process.

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Last nameNameLaboratoryExt.*e-mailProfessional category
Berenguer CarlosJosé1084498jberenguer(at)cbm.csic.esCatedrático Universidad, GA
Mencia CaballeroMario1084664mmencia(at)cbm.csic.esProfesor Contratado Universidad, GA
Pedro MontalbánMiguel Angel de114.34496madepedro(at)cbm.csic.esE. Investigadores Científicos de Organismos Públicos
Sánchez CostaMercedes1084525mercedes.sanchez(at)cbm.csic.esTitulado Sup.de Actividades Técn. y Profes. GP1
Verdu CanoCarlos1084525Titulado Sup.de Actividades Técn. y Profes. GP1

Relevant publications:

  • Baquedano I, Mencía M, Blesa A, Burrus V, and Berenguer J  (2020) ICETh1 & ICETh2, two interdependent mobile genetic elements in Thermus thermophilus transjugation. Environmental Microbiology. 22:158-169. doi: 10.1111/1462-2920.14833. The first integrative and conjugative element for T. thermophilus, which codes for the transjugation DNA donor machinery, is described along with its dependence on the integrase/excisionase module of a second integrative element.
  • Verdú C, Sanchez E. Ortega C, Hidalgo A, Berenguer J, Mencía M. 2019. A modular vector toolkit with a tailored set of thermosensors to regulate gene expression in Thermus. ACS Omega. 4: 14626−14632. A set of modular shuttle plasmids are developed that includes thermosensors that limit the expression in relation to the temperature.
  • García-Quintans N, Bowden L, Berenguer J and Mencía. 2019. DNA interference by a mesophilic Argonaute protein, CbcAgo.  F1000Research 8: 321. https://doi.org/10.12688/f1000research.18445.1) A mesophilic Argonaute protein is purified and described as tool capable to cut at 37ºC  DNA at specific sites selected by synthetic ssDNA guides that will likely be the next generation gene edition tools.
  • Chahlafi, Z., Alvarez, L., Cava, F., Berenguer J. 2018. The role of conserved proteins DrpA and DrpB in nitrate respiration of Thermus thermophilus. Environmental Microbiology 20: 3851-3861. A new mechanism for the detection of nitrate was described for Thermus spp that has nothing to do with the NarX/L two component systems used by most Bacteria.
  • Blesa A., Baquedano I., Quintáns N.G., Mata C.P., Castón J.R., Berenguer J. 2017. The transjugation machinery of Thermus thermophilus: Identification of TdtA, an ATPase involved in DNA donation. PloS Genetics. 13 (3) e1006669. A DNA translocase is identified as the major element required for DNA donation in transjugation in T. thermophilus.
  • Alvarez, L., Quintáns N.G., Blesa A., Baquedano I., Bricio C., Mencía M., Berenguer J. 2017. Hierarchical control of nitrite respiration by transcription factors encoded within mobile gene clusters of Thermus thermophilus. Genes 8: 361. A new sensory system for NO is described encoded by the cluster for nitrite respiration of denitrifying strains of T. thermophilus. The system seems more related to that found in Actynobacteria than to those of Proteobacteria.
  • Blesa A., Quintáns N.G., Baquedano I., Mata C.P., Castón J.R., Berenguer J. 2017. Role of archaeal HerA protein in the biology of the bacterium Thermus thermophilus. Genes  8: 130. A hexameric DNA-helicase of the archaeal HerA family  is described which presence in donor cells is needed for transjugation, likely by providing the ability to repair the damage generated during DNA donation from multiple points in the genome.  
  • Blesa A, César CE, Averhoff B, Berenguer J. 2015. Noncanonical cell-to-cell DNA transfer in Thermus spp. is insensitive to argonaute-mediated interference. J Bacteriol. 197:138-146. The first description of transjugation and its scape to the surveillance Argonaute system.
  • Daan C. Swarts, Matthijs M. Jore, Edze R. Westra, Yifan Zhu, Jorijn H. Janssen, Ambrosius P. Snijders, Yanli Wang,  Dinshaw J. Patel, José Berenguer, Stan J. J. Brouns, John van der Oost. 2014. DNA-guided DNA interference by a prokaryotic Argonaute. Nature 507: 258-261. The pAgo from T. thermophilus constitutes a barrier against external DNA acquired by transformation through a DNA-DNA interference mechanism.
  • Schurig-Briccio LA, Venkatakrishnan P, Hemp J, Bricio C, Berenguer J, Gennis RB. 2013. Characterization of the nitric oxide reductase from Thermus thermophilus. PNAS USA 110: 12613-18. Structure-functional study of the nitric oxide reductases of T. thermophilus showing the absence of a proton channel.
  • Laura Álvarez, Carlos Bricio,  Manuel José Gómez, and José Berenguer. 2011. Lateral transfer of the denitrification pathway among Thermus thermophilus strains. App Environ Microbiol. 77: 1352 -1358.   The HGT transfer of the denitrification capability from a facultative anaerobe strain of T. thermophilus to an aerobic one is described.

Doctoral theses:

  • Alba Blesa Esteban (2016) Horizontal gene transfer in Thermus thermophilus: mechanisms and barriers. Universidad Autónoma de Madrid. Director: José Berenguer.
  • Yamal Al-ramahi González (2013) Ingeniería de proteínas fluorescentes y aplicaciones de localización celular en microorganismos termófilos. Universidad Autónoma de Madrid. Directores: José Berenguer y Aurelio Hidalgo.
  • Noé R. Rivera (2013) Termoestabilización de proteínas de interés biológico. Universidad Autónoma de Madrid. Directores: José Berenguer y Aurelio Hidalgo.
  • Laura Álvarez Muñoz (2012) Análisis de la respiración de nitrito en Thermus thermophilus. Universidad Autónoma de Madrid. Director: José Berenguer.

Funded projects in the last years:

  • INAGOMICS: DNA-DNA interference mediated by the Argonaute protein of Thermus thermophilus and applications in genome edition. BIO2016-77031-R. Period: 01/01/2017 to 30/6/2020. José Berenguer (IP).
  • METAFLUIDICS: Advanced toolbox for rapid and cost-effective functional metagenomic screening: microbiology meets microfluidics. Funding: European Union H2020. GA 685474. Period: 01/06/2016 to 30/05/2020. Coordinator: A. Hidalgo.
  • HOTDROPS: Ultrahigh-throughput platform for the screening of thermostable proteins by thermophilic in vitro transcription-translation and microfluidics. Funding: European Union FP7. GA 324439. Period: 01/06/2013 to 30/05/2017. Coordinator: J. Berenguer.

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