Tuesday, 10th December 2019

Biotechnology and genetics of extreme thermophiles



José Berenguer Carlos




Scientific abstract:

The main aim of our group is focused on the use of thermophiles as biological models and as tools and source of thermozymes for biotechnology. Our laboratory model is Thermus thermophilus (Tth) a thermophilic bacterium of ancestral phylogenetic origin that shows fast growth under laboratory conditions, and highly efficient natural competence apparatus (NCA).


figure 1

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.


As biological models, we have focussed our attention on a mechanism of horizontal gene transfer (HGT) between Tth strains that requires cell to cell contacts and depends on an active NCA in the recipient cell. The mechanism, named transjugation, also needs the presence in the donor cell of the a DNA translocase (TdtA) encoded along a restriction system and a nuclease in an operon belonging to a small Conjugative and integrative Element (ICEth1), which mechanism of transference is currently under study. Another point of attention in this basic biology studies is the role as barrier against HGT played by a homologue to human Argonaute (ThAgo). We know that ThAgo inhibits the entry of extracellular DNA through the NCA by a DNA-DNA interference mechanism. As this barrier action of ThAgo is bypassed when the DNA is transferred by transjugation, a mechanism of activation/deactivation has been hypothesized which is also the subject of our current research. In this context, TthAgo seems to distinguish between potentially dangerous environmental DNA from “trustworthy” DNA obtained from a related strain. Also, as nucleic acid interference constitutes the bases for gene edition, we are working on the use of this and other prokaryotic Ago for this purpose.


figure 2

Figure 2: The use of microdroplets in high-throughput screenings. A) Growth of microorganisms and mammalian cells. B) Screening for recombinant thermozymes expressed in Tth. C) In vitro expression of thermozymes using in vitro transcription/translation derived from Tth. D) Digital PCR to detect rare microorganisms (dark biological matter) and genes. Figure credits: M. Almendros, A. Lopes and M. Sánchez.


A major effort in our group is focused in the discovery of thermostable enzymes (thermozymes) and thermostable variants of enzymes that could better respond to the requirements for industrial biocatalysis. For this, we have used Tth as host for the in vivo selection of thermostable enzymes in large libraries of man-made diversity using newly-developed tools and strains. In parallel, we have incorporated to our laboratory methods of ultrahigh- throughput enzyme screening based on the use of water-in-oil emulsions or microdroplets. The reduction of the test tube to a compartment of picoliters enables performing up to 106 enzyme assays per day and paves the way for future single-cell biology experiments. Microdroplets are generated using self-made silicone microfluidic devices and screened on-chip using a bespoke FACS-like sorter.



Selected articles from the last 4 years:

  • Baquedano I, Mencía M, Blesa A, Burrus V and Berenguer J (2019) ICETh1 & ICETh2, two interdependent mobile genetic elements in Thermus thermophilus transjugation. Environ Microbiol doi: 10.1111/1462-2920.14833
  • Mate DM, Rivera NR, Sanchez-Freire E, Ayala JA, Berenguer J and Hidalgo A (2019) Thermostability enhancement of thePseudomonas fluorescensesterase I by in vivo folding selection inThermus thermophilus. Biotech. Bioeng. doi: 10.1002/bit.27170
  • Verdú C, Sanchez-Freire E, Ortega C, Hidalgo A, Berenguer J* and Mencía M* (2019) A modular vector toolkit with a tailored set of thermosensors to regulate gene expression in Thermus thermophilus. ACS Omegadoi: 10.1021/acsomega.9b02107
  • Alvarez L, Sanchez-Hevia D, Sánchez M and Berenguer J (2018) A new family of nitrate/nitrite transporters involved in denitrification. Int. Microbiol. 22, 19–28. doi: 10.1007/s10123-018-0023-0
  • Chahlafi Z, Alvarez L, Cava F and Berenguer J (2018) The role of conserved proteins DrpA and DrpB in nitrate respiration of Thermus thermophilus. Environ. Microbiol. 20, 3851- 3861. doi: 10.1111/1462-2920.14400
  • Antonucci I, Gallo G, Limauro D, Contursi P, Ribeiro AL, Blesa A, Berenguer J, Bartolucci S, and Fiorentino G (2018) Characterization of a promiscuous cadmium and arsenic resistance mechanisms in Thermus thermophilus HB27 and potential application of a novel bioreporter system. Microb. Cell Fact. 17, 78- 87. doi: 10.1186/s12934-018-0918-7
  • Blesa A, Averhoff B and Berenguer J (2018) Horizontal gene transfer in Thermus sp. Curr. Issues Mol. Biol. 29, 23-36. doi: 10.21775/cimb.029.023
  • Consolati T, Bolívar JM, Petrasek Z, Berenguer J, Hidalgo A, Guisán JM and Nidetzky B (2018) Bio-based pH internally sensitive materials: immobilized yellow fluorescent protein as optical sensor for spatiotemporal mapping of pH inside porous matrices. ACS Appl. Mater. Interfaces 10, 6858-6868. doi:10.1021/acsami.7b16639
  • Antonucci I, Gallo G, Limauro D, Contursi P, Ribeiro AL, Blesa A, Berenguer J, Bartolucci S and Fiorentino G (2017) An ArsR/SmtB family member regulates arsenic resistance genes unusually arranged in Thermus thermophilus HB27. Microb. Biotechnol. 10, 1690-1701. doi: 10.1111/1751-7915.12761
  • Alvarez L, García-Quintáns N, Blesa A, Baquedano I, Bricio C, Mencía M and Berenguer J (2017) Hierarchical control of nitrite respiration by transcription factors encoded within mobile gene clusters of Thermus thermophilus. Genes 8, 361. doi:10.3390/genes812036
  • Ribeiro AL, Sánchez M, Hidalgo A and Berenguer J (2017) Stabilization of enzymes by using thermophiles. Methods Mol. Biol. 1465, 297-312. doi: 10.1007/978-1-4939-7183-1_21
  • Blesa A, Quintáns NG, Baquedano I, Mata CP, Castón JR and Berenguer J (2017) Role of archaeal HerA protein in the biology of the bacterium Thermus thermophilus. Genes 8, 130. doi:10.3390/genes8050130
  • Blesa A, Baquedano I, García-Quintáns N, Mata CP, Castón JR and Berenguer J (2017) The transjugation machinery of Thermus thermophilus: Identification of TdtA, an ATPase involved in DNA donation. PloS Genetics 13, e1006669. doi: 10.1371/journal.pgen.1006669
  • Berenguer J, Mencía M and Hidalgo A (2017) Are in vivo selections on the path to extinction? Microb. Biotechnol. 10, 46-49. doi: 10.1111/1751-7915.1249



Last 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) Engineering of fluorescent proteins and applications in cellular localization studies in thermophilic microorganisms. Universidad Autónoma de Madrid. Directors: José Berenguer and Aurelio Hidalgo.
  • Noé R. Rivera (2013) Thermal stabilization of proteins with biotechnological interest. Universidad Autónoma de Madrid. Directors: José Berenguer and Aurelio Hidalgo.
  • Laura Alvárez Múñoz (2012) Analysis of nitrite respiration in Thermus thermophilus. Universidad Autónoma de Madrid. Director: José Berenguer.



International Projects and Excellence networks:

  • 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.
  • CARBAZYMES: Sustainable industrial processes based on a C-C bond-forming enzyme platform. Funding: European Union H2020. GA 635595. Period: 01/04/2015 to 31/03/2019. Role: Partner.
  • RedEx: National Network on Extremophilic Microorganisms. National project on excellence networks. BIO2015-71815. Period: 01/01/2016 to 30/06/2018. Role: Partner.
  • 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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