Analysis of CRISPR–Cas loci distribution in Xanthomonas citri and its possible control by the quorum sensing system

Abstract

Xanthomonas is an important genus of plant-associated bacteria that causes significant yield losses of economically important crops worldwide. Different approaches have assessed genetic diversity and evolutionary interrelationships among the Xanthomonas species. However, information from clustered regularly interspaced short palindromic repeats (CRISPR) has yet to be explored. In this work, we analyzed the architecture of CRISPR-Cas loci and presented a sequence similarity-based clustering of conserved Cas proteins in different species of Xanthomonas. Although absent in many investigated genomes, Xanthomonas harbors subtype I-C and I-F CRISPR-Cas systems. The most represented species, Xanthomonas citri, presents a great diversity of genome sequences with an uneven distribution of CRISPR-Cas systems among the subspecies/pathovars. Only Xanthomonas citri subsp. citri and Xanthomonas citri pv. punicae have these systems, exclusively of subtype I-C system. Moreover, the most likely targets of the X. citri CRISPR spacers are viruses (phages). At the same time, few are plasmids, indicating that CRISPR/Cas system is possibly a mechanism to control the invasion of foreign DNA. We also showed in X. citri susbp. citri that the cas genes are regulated by the diffusible signal factor (DSF), the quorum sensing (QS) signal molecule, according to cell density increases, and under environmental stress like starvation. These results suggest that the regulation of CRISPR-Cas by QS occurs to activate the gene expression only during phage infection or due to environmental stresses, avoiding a possible reduction in fitness. Although more studies are needed, CRISPR-Cas systems may have been selected in the Xanthomonas genus throughout evolution, according to the cost-benefit of protecting against biological threats and fitness maintenance in challenging conditions.