# Inactivation of Cysteine Synthase CysK-A enhances flocculation, biofilm formation, and sensitivity to oxidative stress in Azospirillum brasilense Sp7

## TL;DR

This study shows that inactivating the CysK-A enzyme in Azospirillum brasilense Sp7 affects growth, stress tolerance, and aggregation behaviors.

## Contribution

The study reveals the multifaceted role of CysK-A in regulating aggregative behaviors and stress responses in A. brasilense Sp7.

## Key findings

- Inactivation of CysK-A leads to growth defects and increased flocculation and biofilm formation.
- The cysK-A mutant shows increased sensitivity to oxidative stress and higher adhesion to radish seeds.
- CysK-A inactivation correlates with intracellular accumulation of cyclic-di-GMP under stress conditions.

## Abstract

Cysteine biosynthesis is a critical metabolic pathway for bacterial physiology. However, the full impact on the lifestyle of the plant-beneficial bacterium Azospirillum brasilense Sp7 is not completely understood. Our previous work identified a cysteine synthase A (CysK-A) as a key enzyme in cysteine synthesis in A. brasilense Sp7, but its inactivation did not lead to cysteine auxotrophy, suggesting functional redundancy in this type of enzyme. Here, we comprehensively characterized an A. brasilense AR cysK-A mutant, revealing a multifaceted phenotype that highlights the indispensable role of CysK-A. The cysK-A mutant exhibited a growth defect that was rescued by genetic and chemical complementation, underscoring the importance of de novo cysteine synthesis for optimal metabolic fitness. Furthermore, the cysK-A mutant displayed a striking hyper-aggregative behavior, characterized by significantly enhanced flocculation, biofilm formation, and exopolysaccharide production. Confocal microscopy revealed an abundance of ovoid, cyst-like cells. This transition toward a sessile lifestyle, induced by cysteine limitation stress, correlated with the intracellular accumulation of cyclic-di-GMP, as determined by a c-di-GMP biosensor assay. Additionally, the cysK-A mutant exhibited increased sensitivity to exogenous hydrogen peroxide stress, a deficiency that was restored by cysK-A complementation. The mutation also led to enhanced adhesion to radish seeds; however, it did not result in statistically significant differences in overall radish seedling colonization after seven days, possibly due to compensatory mechanisms. Collectively, our findings establish CysK-A as crucial for optimal growth, stress tolerance, and the regulation of aggregative behaviors in A. brasilense Sp7, providing insights into the adaptive strategies employed by this important plant-associating bacterium.