Exploración de los mecanismos de adaptación y resistencia a radiación UV en microorganismos halófilos extremos

Abstract

The objective of this doctoral thesis was to explore the mechanisms of resistance and adaptation to ultraviolet B radiation (UVB) in extreme halophiles. On one hand, to isolate genes involved in radiation resistance from these microorganisms, we used a culture-independent technique, functional metagenomics. For this, two metagenomic libraries were constructed, using Escherichia coli as host, with DNA (metagenome) extracted from brines of two crystallizing ponds from "Bras del Port" saltern (Santa Pola, Alicante), called CR30 and CCAB (39% and 30% salt respectively). Eight UVB resistant clones were isolated by functional screening of metagenomic libraries. In the eight environmental DNA fragments recovered, fifteen bacteria and archaea genes responsible for the phenotype of UVB resistance were identified. These genes encode for proteins previously identified in DNA damage resistance, others with known function but not related to radiation resistance, and several proteins with unknown function, similar to families of hypothetical proteins. Many of the genes also showed resistance to 4-NQO, a compound that mimics the DNA damage produced by UVB radiation, so these genes could be related to DNA protection or repair; furthermore, some of them also showed resistance to the toxic compound perchlorate. On the other hand, to understand how the cellular machinery of extreme halophiles responds and adapts to UVB radiation, a transcriptomic study of the bacteria Salinibacter ruber and the archaea Haloquadratum walsbyi was carried out, in order to analyze the changes in gene expression after being exposed to a sublethal dose of UVB radiation and recovered in the presence of light or darkness for Sal. ruber, and only light for Hqr. walsbyi. In both microorganisms, the differential expression of less than 20% of the genes was demonstrated. Both in Sal. ruber and Hqr. walsbyi genes involved in DNA replication and cell division are repressed, and consistently, also genes involved in transcription, protein translation, and metabolic pathways that provide energy and molecules needed for cell growth and division (with some differences in Sal. ruber recovered in light, in which the glyoxylate pathway and gluconeogenesis are induced). The repression of replication would allow the repair of DNA damage caused by UVB radiation, avoiding the division of mutated cells. In both microorganisms is taking place the induction of genes related to: i) DNA repair and protection (exonucleases, endonucleases, Dps); ii) protection, repair of oxidative damage and protein degradation (chaperones, thioredoxins, glutaredoxins and proteases); and iii) antioxidants (superoxide dismutase, glutathione) to eliminate reactive oxygen species generated by UVB. On the other hand, possibly to get the cells to move away from radiation, genes involved in the formation of gas vesicles in Hqr walsby are repressed, which allow them to float in the medium, and the motility genes of Sal. ruber are induced.