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

Cysteine biosynthesis is a critical metabolic pathway for bacterial physiology. However, the full impact on the lifestyle of the plant-beneficial bacterium <i>Azospirillum brasilense</i> Sp7 is not completely understood. Our previous work identified a cysteine synthase A (CysK-A) as a key enzyme in cysteine synthesis in <i>A. brasilense</i> Sp7, but its inactivation did not lead to cysteine auxotrophy, suggesting functional redundancy in this type of enzyme. Here, we comprehensively characterized an <i>A. brasilense</i> AR <i>cysK</i>-A mutant, revealing a multifaceted phenotype that highlights the indispensable role of CysK-A. The <i>cysK</i>-A mutant exhibited a growth defect that was rescued by genetic and chemical complementation, underscoring the importance of <i>de novo</i> cysteine synthesis for optimal metabolic fitness. Furthermore, the <i>cysK</i>-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 <i>cysK</i>-A mutant exhibited increased sensitivity to exogenous hydrogen peroxide stress, a deficiency that was restored by <i>cysK</i>-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 <i>A. brasilense</i> Sp7, providing insights into the adaptive strategies employed by this important plant-associating bacterium.