The Reverse Transcriptases associated with CRISPR-Cas systemsPhylogenetic relationships and functional characterization

Abstract

The objective of this thesis consisted of the characterization of novel groups of RTs linked to CRISPR-Cas systems. An initial analysis of the different distribution of RTs in bacteria and archaea indicates that specific prokaryotic groups have recruited particular types of RTs in order to improve their responses in the environmental conditions of their ecological niche. Then, further analysis focused on the RTs related to CRISPR-Cas systems clustering them in 15 phylogenetic clades which at least present three evolutionary origins supporting a “multiple origins” model. These RTs are found alone, fused to the C-termini of the Cas1 domain (RTCas1) or as a multidomain protein (Cas6RTCas1). Furthermore, most RTs are associated with type III CRISPR-Cas systems, whereas a few examples are related to types I-A and VI. The first criterion for the functional characterization of RT-containing CRISPRCas systems was to determine the exogenous RT activity in vitro of several RT homologs. Then, the study focused on two systems: the adaptive operon from the Cyanobacterium Scytonema hofmanni PCC 7110, which harbor an RT alone, and that from the Proteobacterium Vibrio vulnificus YJ016, that contain an RTCas1 fusion protein. In both systems, the purification of the different proteins of the adaptive operon allows to study their interactions, demonstrating that the two different V. vulnificus Cas2 proteins, Cas2A and Cas2B, form a stable heterodimer complex. In vivo assays revealed that the RTCas1-Cas2A-Cas2B adaptive operon from V. vulnificus YJ016 is able to acquire spacers in a heterologous host (E. coli) in a process in which the two different Cas2 are required. Moreover, mutation in the RT active site strongly impaired novel acquisition events. It has been also demonstrated that this system is able to acquire spacers directly from RNA. In addition, the analysis of the acquired spacers and their sequence revealed particular features that may be related to a specific recognition by the adaptive operon. Finally, assays in the natural host have confirmed that the V. vulnificus type III-D CRISPR-Cas interference module is functional as long as their DNA target is transcriptionally active.