# Deciphering strain differences in codY regulation of Clostridioides difficiles sporulation

## TL;DR

This study explores how CodY, a nutrient-sensing regulator, affects spore formation in two strains of Clostridioides difficile, revealing strain-specific differences in gene regulation.

## Contribution

The study identifies CodY-regulated factors influencing sporulation and highlights differences in CodY regulation between two C. difficile strains.

## Key findings

- CodY suppresses premature spore formation in both 630∆erm and UK1 strains.
- CodY-dependent gene expression differs between the 630∆erm and UK1 strains.
- CRISPRi knockdowns revealed multiple CodY-regulated factors impacting sporulation.

## Abstract

Clostridioides difficile is an anaerobic, spore-forming pathogen that causes diarrhea, colitis, and even death. C. difficile grows and replicates in the intestine as a vegetative bacillus, but must transition into a dormant spore to survive and transmit in the environment. The transformation into a spore is a complex developmental process that is regulated in response to conditions within the host, most notably nutrient limitation. Nutrient availability is sensed by C. difficile through transcriptional regulators, such as CodY. CodY is a global nutritional gene regulator that controls gene expression in response to branched-chain amino acids (BCAAs) and guanosine-triphosphate (GTP). It was previously observed that CodY represses C. difficile sporulation, but the impact of CodY on sporulation has differed considerably by strain. Here, we investigated the effects of CodY on gene expression during sporulation in the two common research strains 630∆erm (ribotype 012) and UK1 (ribotype 027). We confirmed that CodY suppressed premature spore formation in both strains through time-elapsed sporulation assays with codY mutants. Through transcriptional analyses of codY mutant sporulation, we defined the similarities and differences in the CodY-dependent gene expression between strains. We also identified differences in putative CodY sites within the 630 and UK1 genomes that may influence CodY regulation. Finally, we performed CRISPRi knockdowns to examine the effects of selected CodY-regulated genes, demonstrating the impact of multiple CodY-dependent factors on sporulation.

Clostridioides difficile spore formation is crucial for transmission and survival of the bacterium. Spore formation is triggered by the availability of crucial nutrients, which CodY and other regulators sense. However, the mechanism by which CodY represses sporulation in C. difficile is poorly understood. In this study, we identified several CodY-regulated factors that could play a role in sporulation both in 630∆erm and UK1 strains. Our results show that many factors under the regulation of CodY can impact sporulation.