# Orn-mediated c-di-GMP regulates the CRISPR-Cas system to confer stress response in Mycobacterium tuberculosis

## TL;DR

This study reveals how the CRISPR-Cas system in Mycobacterium tuberculosis helps the bacteria respond to stress through a regulatory pathway involving c-di-GMP and the Orn protein.

## Contribution

The study identifies a novel regulatory mechanism where Orn-mediated c-di-GMP controls the CRISPR-Cas system to enhance stress response in Mtb.

## Key findings

- The Mtb CRISPR-Cas system mitigates oxidative stress and antibiotic treatment via the Cas10 HD domain.
- Orn regulates the CRISPR-Cas system by modulating c-di-GMP levels, which in turn affects the transcriptional regulator Rv3058.
- This regulatory pathway activates protective mechanisms like DNA repair and cell envelope maintenance.

## Abstract

Mycobacterium tuberculosis (Mtb) possesses a type III-A CRISPR-Cas system and has anti-plasmid immune activity. However, whether this system exerts other additional functions remains to be characterized. Here, we investigated the in vivo roles of the Mtb CRISPR-Cas system. We show that this system is transcriptionally dependent and exhibits limited ability to counteract exogenous nucleic acids, primarily through the Csm6 protein rather than the Cas10 HD domain. We further demonstrate that this system plays a role in mitigating oxidative stress and antibiotic treatment, a function mainly mediated by the Cas10 HD domain. Importantly, through transposon library screening, we identified oligoribonuclease (Orn) as a regulatory protein of the Mtb CRISPR-Cas system. Deletion of the orn gene resulted in elevated c-di-GMP levels. A subsequent biotin-labeled c-di-GMP pull-down assay identified the transcriptional regulator Rv3058. Knockdown of rv3058 significantly increased cas6 promoter activity, and its transcriptional repressor function was directly modulated by c-di-GMP. This regulatory pathway enhances stress defense by activating multiple protective pathways, including DNA repair, cell envelope maintenance, and iron homeostasis regulation. Together, we conclude that the regulation of the CRISPR-Cas system by Orn-mediated c-di-GMP contributes to oxidative and antibiotic stress responses in Mtb.

Graphical Abstract